Chondrogenic progenitor cells, protocol for derivation of cells and uses thereof

ABSTRACT

The present invention provides an isolated population of chondrocyte precursor cells wherein 1% or less of the cells express Oct4, Nanog and/or TRA-1-60, 7% or less of the cells express no collagen II, collagen X, CD105 or Stro-1 and 85% or more of the cells express CBFA1, methods for preparing such cells and uses of chondrocyte cells derived from said precursor cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Appln. No.61/321,982, filed on Apr. 8, 2010, which is incorporated herein byreference.

TECHNICAL FIELD

The present invention relates generally to the fields of cell biology,embryonic stem cells, and cell differentiation. The invention discloseschondrogenic progenitor cells and a method for the preparation of suchcells and fully differentiated chondrocytes, including uses of thecells.

BACKGROUND

Chondrocytes are specialised cells found in cartilage. Chondrocytes incartilage produce a large amount of extracellular matrix which iscomposed of collagen fibers, ground substance, which is rich inproteoglycan, and elastin fibers. The cartilage tissue performs astructural and mechanical function in skeletal joints and any disease orinjury is consequently debilitating for patients.

Cartilage degradation is a hallmark of two disease groups:osteoarthritis, a degenerative condition, and rheumatoid arthritis,which is primarily caused by inflammation. The degradation leads tojoint pain and mobility impairment to a degree that can be disabling.

Considerable progress has been made in the development ofanti-inflammatory agents effective for inhibiting the progress of suchdiseases. However, where degradation or injury has already taken place,new therapies are needed to assist in the regenerating of jointcartilage.

In the field of regenerative medicine, efforts have been directed atdeveloping cell populations capable of repairing cartilage. Establishedlines of articular chondrocytes have been described in WO 96/18728 andmethods for chondrocyte growth and differentiation have been reported inWO 98/55594. WO 00/27996 reports serum-free medium for chondrocyte likecells, comprising minimum essential medium, growth factors, lipids andamino acids. U.S. Pat. No. 6,150,163 outlines chondrocyte mediaformulations and culture procedures, in which dedifferentiated humanarticular chondrocytes are grown in a medium containing TGFβ and eitherinsulin or insulin-like growth factor. It has also been reported thatprimary chondrocytes cultured in vitro will de-differentiate if nottreated with the appropriate factors (Benya et al. Cell 1982 30:215).The cells that are produced are fibroblastic in appearance and may beMSC-like.

Jorgensen et al. (Ann. Rheum. Dis. 60:305, 2001) reviews recent progressin stem cells for repair of cartilage and bone in arthritis. Jakob etal. (J. Cell. Biochem. 26:81, 2001) studied specific growth factorsinvolved in expansion and redifferentiation of adult human articularchondrocytes that enhance chondrogenesis and cartilage formation. M.Brittberg (Clin. Orthop. 367 Suppl:S147, 1999) reviews currentchondrocyte transplantation procedures in which pure chondrocytes orother mesenchymal cells are harvested autologously or as allografts froma healthy tissue source, expanded in vitro, and then implanted into thedefect at high density.

Despite the initial success of the clinical methods reported to date, itis clear that current sources of chondrocytes are inadequate to treatmost of the instances of cartilage degeneration that present themselvesat the clinic. In addition, problems concerning the use ofimmunosuppressive drugs have complicated the success of developing newtransplantation protocols.

Regenerative medicine is also benefiting from recent advances relatingto the isolation, culture, and use of various types of progenitor cells.Embryonic stem cells have two very special properties: First, unlikeother typical mammalian cell types, they can be propagated in culturealmost indefinitely while maintaining their pluripotency, providing avirtually unlimited supply. Second, they can be used to generate avariety of tissue types of interest as a source of replacement cells andtissues for use in tissue therapy, or for use in the screening ofpharmaceutical agents. Consequently, stem cells are seen as possiblesources of chondrocytes.

Kramer et al. (Mech. Dev. 92:193, 2000) reported that mouse embryonicstem cells can be modulated with bone morphogenic proteins (BMP-2 andBMP-4) to produce cells that stained with Alcian blue, a feature ofchondrocytes, and expressing the transcription factor scleraxis.However, the mouse model of embryonic stem cell development does notnecessarily yield strategies for differentiation that are applicable toother species (see, e.g. Ginis et al. (2004) Dev. Biol 269:360).

Thomson et al. (U.S. Pat. No. 5,843,780; Proc. Natl. Acad. Sci. USA92:7844, 1995) were the first to successfully isolate and propagatepluripotent stem cells from primates. They subsequently establishedhuman embryonic stem (hES) cell lines from human blastocysts (Science282:114, 1998). Gearhart and co-workers established human embryonic germ(hEG) cell lines from fetal gonadal tissue (Shamblott et al., Proc.Natl. Acad. Sci. USA 95:13726, 1998; and U.S. Pat. No. 6,090,622). BothhES and hEG cells have the long-sought characteristics of pluripotentstem cells: they can be cultured extensively without differentiating,they have a normal karyotype, and they are capable of producing a numberof important cell types including cell types from all three primary germlayers.

Mesenchymal progenitors can be generated from hES cells according to themethod described in WO 03/004605. The hES-derived mesenchymal cells canthen be further differentiated into osteoblast lineage cells in a mediumcontaining an osteogenic factor, such as bone morphogenic protein(particularly BMP-4), a ligand for a human TGF-β receptor, or a ligandfor a human vitamin D receptor (WO 03/004605; Sotile et al., CloningStem Cells 2003; 5(2):149-55). Chondrocytes or their progenitors can begenerated by culturing hES cells in microaggregates with effectivecombinations of differentiation factors listed in WO 03/050250.

Hegert et al. (J. Cell Sci. 115:4617, 2002) reported the differentiationplasticity of chondrocytes derived from embryoid bodies composed ofmouse embryonic stem cells. Toh et al. describes differentiation andenrichment of expandable chondrogenic cells from human embryonic stemcells in vitro (J. Cell. Mol. Med., 2009—early online publicationcitation reference 10.1111/j.1582-4934.2009.00762.x).

There remains a need for efficient, scaleable methods capable ofproducing sufficient quantities of chondrocyte lineage cells, includingboth chondrocyte progenitors as well as mature chondrocytes fortherapeutic and research applications. It would also be useful if astable chondrocyte precursor could be isolated that could be maintainedin culture under scaleable conditions and which could readily bedifferentiated into mature chondrocytes or cells expressing proteins,such as collagen II, aggrecan and glycosaminoglycans.

However, even with the preparation of chondrocytes derived fromundifferentiated progenitor cell populations, such as hES cells, thereremains the need to use immunosuppressive drugs in transplantationtherapies using chondrocyte cell populations which is unfavourable forthe long term success of transplants in patients.

Accordingly, it is necessary to develop a new approach to differentiateprimate pluripotent cells into fully functional chondrocytes which avoidthe need to use immunosuppressant drugs in transplantation as well asthe chondrocyte precursor cells types which could readily give rise tosuch chondrocytes. It is also necessary to develop a new approach totherapeutically using chondrocytes, such as chondrocytes differentiatedin vitro from primate pluripotent stem cells, without the use ofimmunosuppressive and/or anti-inflammatory agents. It is furthernecessary to develop a new approach to treating subjects withchondrocytes, such as chondrocytes differentiated in vitro from primatepluripotent stem cells, without the use of immunosuppressive and/oranti-inflammatory agents.

SUMMARY OF THE INVENTION

The present invention provides a novel population of chondrocyteprogenitor or precursor cells which are characterised by the expressionof certain protein markers as defined herein. The cells may also becharacterised by the loss of transdifferentiation potential

The population of chondrocyte precursors or progenitors can be obtainedby differentiating pPS cells by a method of the present invention, andis capable of forming progeny having the characteristics of maturechondrocytes. The chondrocyte progenitors are no longer pluripotent, butare committed to the chondrocyte development pathway

The invention also provides a system as defined herein for thepreparation of chondrocytes and chondrocyte progenitor or chondrocyteprecursor cells. The isolated or in vitro population of chondrocyteprogenitor or chondrocyte precursor cells is prepared by differentiatinga population of primate pluripotent stem (pPS) cells in chondrogenicmedia until the cells are greater than 75% confluent after which thecells are washed and resuspended in a defined minimal growth media andcultured for a further period until the cells are differentiated into apopulation of cells which are characterised by the loss oftransdifferentiation potential, e.g. the cells cannot be subsequentlycultured in osteogenic media to form osteoblasts. The cells of thisculture are also characterised by a fibroblast morphology, the absenceof expression of ES pluripotency markers or mesenchymal stem cell (MSC)markers, and the absence of expression of chondrogenesis markers. Thecells of this culture are also characterised by the presence ofexpression of nuclear markers for hypertrophic chondrocytes andosteogenesis. This feature therefore distinguishes the cells fromprimary (non-hypertrophic) chondrocytes.

Suitable assays to detect these markers are described herein.Pluripotency markers include Oct4, Nanog and/or TRA-1-60. Markers ofchondrogenesis include collagen II. Nuclear markers for hypertrophicchondrocytes and osteogenesis include CBFA1/RunX2.

The loss of transdifferentiation potential, e.g., that the cells cannotbe subsequently cultured in osteogenic media to form osteoblasts, can beshown by the absence of mineralisation in the chondrocyte precursorcells upon further culture in osteogenic media.

Characterisation by expression markers of chondrocyte progenitor orchondrocyte precursor cells prepared according to a method of theinvention is shown in Table 1, with an absence of expression(“negative”) being indicated thus “−” and the presence of expression(“positive”) being indicated thus “+”:

TABLE 1 Expression status Marker (−/+) Characteristic Nanog −pluripotency Oct4 − pluripotency Tra-1-60 − pluripotency Collagen II lowchondrogenesis CBFA1/RunX2 + hypertrophic chondrocytes/osteogenesisCollagen X − hypertrophic chondrocytes Osteocalcin − osteogenesis CD105− chondrocytes Stro-1 − mesenchymal stem cells

Less than about 7% of the cells in a population express no Collagen II,whereas the remainder 93% of the population express a low level of themarker. Expression of this marker is therefore less than the level ofexpression seen in a population of fully differentiated chondrocytes.

The chondrocyte progenitor or chondrocyte precursor cells aredistinguished from previously known chondrocyte cells or precursor cellsin view of the above properties. The cells may be referred to asdedifferentiated committed chondrocyte progenitor cells (DCCPC) orInduced chondrocyte precursor cells (ICPC) by virtue of the means usedto prepare the cells. In view of the ability of the cells to besubsequently differentiated into chondrocytes, the cells may also bedescribed as “Forward-Back Chondrocytes” or FBCs. Any of these terms maybe used interchangeably.

The DCCPC can be further cultured in chondrogenic media after washing.Subsequent culture yields a population of cells that can becharacterised as chondrocytes using the markers collagen II and collagenX. The chondrocytes produced express high levels of collagen II butlittle or no collagen X.

The present invention also provides populations of chondrocytesdifferentiated from DCCPC prepared according to the present inventionfor use in transplantation. Surprisingly, the fully differentiatedchondrocyte cell populations may be administered to a subject withoutimmuno-suppressive compounds such as FK-506, cyclosporin or the like.Moreover, anti-inflammatory agents such as prednisone and the like arenot required either. This embodiment of the invention extends to amethod of treating a degenerative cartilage disease or cartilage injurycomprising transplanting a population of chondrocyte cells preparedDCCPC of the present invention into a subject in need thereof.

The invention also provides an isolated or in vitro cell populationcontaining chondrocytes, obtained by differentiating the DCCPC describedherein. Chondrocyte lineage cells can be identified by the ability tosynthesize for example Type II collagen or aggrecan from an endogenousgene. Preferably, the population contains a minimal proportion of cellsthat synthesize elastic cartilage, fibrocartilage, hypertrophiccartilage or bone.

The proportion of undifferentiated pluripotent cells in the populationis preferably minimized, and any residual undifferentiated cells are notthe cells responsible for forming the chondrocytes upon furtherproliferation.

Another embodiment of the invention is a method for producing cartilageby incubating a cell population of this invention under conditions whereconnective tissue proteins are produced. Another embodiment of theinvention is a method of screening a compound for its ability tomodulate chondrocyte growth, differentiation, or synthesis of cartilagecomponents, by combining the compound with a cell population of theinvention and determining its effect.

Another embodiment of the invention is a method of differentiating pPScells in vitro into chondrocytes comprising culturing the pPS in a mediasuitable for differentiating the pPS cells into chondrocyte precursorcells (and optionally isolating the chondrocyte precursor cells) andthen changing the culture conditions such that the chondrocyteprecursors de-differentiate into cells with a fibroblast-like morphologyand nuclear CBFA-1 expression, followed by subsequent re-differentiationof the cells into fully differentiated chondrocytes.

Optionally, replication capacity of the stem cells, such as primatepluripotent stem cells or human embryonic stem cells, can be improved byincreasing telomerase activity.

Another embodiment of the invention is a pharmaceutical composition forproducing, repairing, or maintaining cartilage in vivo, containing acell population of this invention or a fully differentiated chondrocytecell population derived therefrom—and the use of such medicaments forreconstructing cartilage in a subject, including articular cartilage,for example, in cosmetic surgery or the treatment of joint trauma,arthritis, or osteoarthritis.

These and other embodiments of the invention are further described asfollows.

DRAWINGS

FIG. 1 shows morphology changes through the DCCPC protocol. After theinitial chondrogenic differentiation for 14 days H7 cells condense intodense three dimensional colonies. A large amount of cell death isobserved and is seen here as phase bright clusters on top of the liveadherent cells (A). After 5 days in the de-differentiation media cellshave migrated out from the three dimensional colonies into the freespace around them to form a monolayer of fibroblast like cells (B).

FIG. 2 shows nuclear CBFA-1 protein expression in DCCPC by CBFA-1/RunX2staining on DCCPC. H7 cells cultured in mTeSR™ or Conditioned Medium(CM) before entering the DCCPC protocol were stained with an anti-CBFA-1antibody. The hESC line RCM1 was also stained with the same antibody. Inall cases the CBFA-1 antibody co-localises with the DNA binding dyeDAPI.

FIG. 3 shows presence of collagen type I and mineralisation in DCCPCdifferentiated with osteogenic media. DCCPC fail to mineralise matrixafter osteogenic differentiation. DCCPC generated from H7 (A) and H1 (B)cell lines show an increase in extracellular collagen type I proteinproduction after culture in osteogenic media. The Von Kossa stainingprotocol will give a dark brown/black stain when calcium deposits arepresent. The absence of staining in both cell lines indicates theabsence of calcium and therefore the absence of matrix mineralisation.

FIG. 4 shows promotion of in vivo cartilage repair by DCCPC after 21days in a WT rat using re-differentiated DCCPC. DCCPC generated from H7line previously cultured in CM were cultured in a construct format andimplanted into WT rats. One construct was placed into a 1 mm defect inthe trochlea groove of the rat hind limb. After 21 days the limb wascryosectioned and stained with H&E. The arrow indicates the extent ofthe regenerated material.

FIG. 5 shows a schematic representation of DCCPC protocol. Usingchondrogenic media pPS are differentiated towards chondroprogenitor-likecells (1). This process is characterised by high cell death, loss ofpluripotency and multipotency markers and the initiation of collagentype II production. Further application of chondrogenic media producesfully differentiated chondrocytes but is not part of the DCCPC protocol(2), instead the chondrocyte precursor cells are incubated withde-differentiation media until the cells show a fibroblast-likemorphology and nuclear CBFA-1 expression. The absence of pluripotencyand multipotency markers is maintained (3). The DCCPC can then bere-differentiated to fully differentiated chondrocytes usingchondrogenic media (4).

DETAILED DESCRIPTION

This invention provides a means to prepare populations of DCCPC withimportant and useful properties. They can be grown and maintained inbulk and then readily differentiated into cells that express collagenII, such as mature chondrocytes. The differentiated chondrocytesprepared from such cells can be used in applications such astransplantation therapy and screening methods as described herein.

The disclosure that follows provides a full description of how to makethe chondrocyte precursor cells of this invention, as well aschondrocytes derived therefrom. It provides extensive illustrations ofhow these cells can be used in research and pharmaceutical development.The disclosure also provides pharmaceutical compositions, devices, andtreatment methods for the use of DCCPC for regeneration and remodellingof cartilage to restore joint mobility and for cosmetic purposes.

DEFINITIONS

For purposes of this disclosure, unless otherwise specified, the term“chondrocyte” refers to mature cells capable of modelling cartilage bythe synthesis of Type II collagen and aggrecan.

The term “dedifferentiated committed chondrocyte progenitor cells(DCCPC)” is used to refer to specialised progenitor cells prepared by amethod of the present invention that can be differentiated to formmature chondrocytes and which are characterized by the marker expressionprofile described infra. Cells of a chondrocyte morphology have arounded-up cell body and in a chondrogenic culture the cells clusterinto dense colonies.

In the context of cell ontogeny, the adjective “differentiated” is arelative term. A “differentiated cell” is a cell that has progressedfurther down the developmental pathway than the cell it is beingcompared with.

A “differentiation agent”, as used in this disclosure, refers to one ofa collection of compounds that are used in culture systems of thisinvention to produce differentiated cells of the chondrocyte lineage(including precursor cells, such as DCCPC and terminally differentiatedcells such as mature chondrocytes). No limitation is intended as to themode of action of the compound. For example, the agent may assist thedifferentiation process by inducing or assisting a change in phenotype,promoting growth of cells with a particular phenotype or retarding thegrowth of others. It may also act as an inhibitor to other factors thatmay be in the medium or synthesized by the cell population that wouldotherwise direct differentiation down the pathway to an unwanted celltype.

Prototype “primate Pluripotent Stem cells” (pPS cells) are pluripotentcells capable under the right conditions of producing progeny of severaldifferent cell types. pPS cells are capable of producing progeny thatare derivatives of each of the three germ layers: endoderm, mesoderm,and ectoderm, according to a standard art-accepted test, such as theability to form a teratoma in a suitable host, or the ability todifferentiate into cells having markers for tissue types of all threegerm layers in culture.

Included in the definition of pPS cells are embryonic cells of varioustypes, exemplified by hES cells, defined below; embryonic stem cellsfrom other primates, such as Rhesus or marmoset stem cells (Thomson etal., Proc. Natl. Acad. Sci. USA 92:7844, 1995; Developmental Biology38:133, 1998); and human embryonic germ (hEG) cells (Shamblott et al.,Proc. Natl. Acad. Sci. USA 95:13726, 1998). Other types of pluripotentcells are also included in the term. Any cells of primate origin thatare capable of producing progeny that are derivatives of all threegerminal layers are included, regardless of whether they were derivedfrom embryonic tissue, fetal tissue, or other sources. It is beneficialto use pPS cells that are karyotypically normal and not derived from amalignant source.

pPS cells include cells and established cell lines. The cells may bederived from pre-embryonic, embryonic, or fetal tissue at any time afterfertilization. as Also included in the term are induced pluripotent stem(iPS) cells, which have the characteristic described above (see, e.g.,Takahashi et al. (2007) Cell 131:1).

Primate pluripotent stem cells typically express the stage-specificembryonic antigens (SSEA) 3 and 4, and markers detectable usingantibodies designated TRA-1-60 and TRA-1-81. Undifferentiated pPS cellsalso typically express the transcription factor Oct 3/4, Cripto,gastrin-releasing peptide (GRP) receptor, podocalyxin-like protein(PODXL), nanog and telomerase reverse transcriptase, e.g., hTERT (US2003/0224411 A1), as detected by RT PCR.

Prototype “human Embryonic Stem cells” (hES cells) are described byThomson et al. (Science 282:1145, 1998; U.S. Pat. No. 6,200,806). Thescope of the term covers pluripotent stem cells that are derived from apre-implantation blastocyst, such as an in vitro fertilized egg, beforesubstantial differentiation of the cells into the three germ layers.Those skilled in the art will appreciate that except where explicitlyrequired otherwise, the term includes primary tissue and establishedlines that bear phenotypic characteristics of hES cells, and derivativesof such lines that still have the capacity of producing progeny of eachof the three germ layers.

pPS cell cultures, such as hES cell cultures are described as“undifferentiated” when a substantial proportion of stem cells and theirderivatives in the population display morphological characteristics ofundifferentiated cells, clearly distinguishing them from differentiatedcells of embryo or adult origin. Undifferentiated pPS cells are easilyrecognized by those skilled in the art, and typically appear in the twodimensions of a microscopic view with high nuclear/cytoplasmic ratiosand prominent nucleoli. It is understood that colonies ofundifferentiated cells within the population will often be surrounded byneighboring cells that are differentiated. Nevertheless, theundifferentiated colonies persist when the population is cultured orpassaged under appropriate conditions, and individual undifferentiatedcells constitute a substantial proportion of the cell population.Cultures that are substantially undifferentiated contain at least 20%undifferentiated pPS cells on an ongoing basis, and may contain at least40%, 60%, or 80% in order of increasing preference (in terms percentageof cells with the same genotype that are undifferentiated).

As discussed above, other sources of pluripotent cells include inducedpluripotent stem (iPS) cells. The iPS cells can be prepared by thetechnique of Yamanaka et al. using retroviral infection of fibroblastswith genes Oct-3/4, SOX2, c-Myc, and Klf4. Selection of iPS cells usingthe marker Nanog appears to be advantageous (Takahashi, K. & Yamanaka,S., Cell, 126:663, 2006; Yamanaka S, et al. Nature 448:313, 2007; WernigM, et al. Nature 448:318, 2007; Maherali N, et al. Cell Stem Cell 1:55,2007). More recently, Thomson et al. used a slightly altered mix ofgenes OCT4, SOX2, NANOG, and a different gene LIN28 using a lentiviralsystem (Science, 318:1917, 2007) and also successfully withnon-integrating episomal vectors (Science, 324:797, 2009).

Whenever a culture or cell population is referred to in this disclosureas proliferating “without differentiation”, what is meant is that afterproliferation, the composition is substantially undifferentiatedaccording to the preceding definition. Populations that proliferatethrough at least four passages (˜20 doublings) without differentiationwill contain substantially the same proportion of undifferentiated cells(or possibly a higher proportion of undifferentiated cells) whenevaluated at the same degree of confluence as the originating culture.

A “nutrient medium” is a medium for culturing cells containing nutrientsthat promote proliferation. The nutrient medium typically containsisotonic saline, buffer, a protein source (in the form of one or moreadded proteins or amino acids), and potentially other exogenously addednutrients and growth factors.

A “chondrogenic medium” is comprised of essential minerals, amino acids,vitamins and substrates for the culture of cells as well as factorsnecessary for differentiating cells into chondrocytes. A typicalchondrogenic medium may be comprised of a defined minimal growth mediumsupplemented by at least one hormone, growth factor, non-essential aminoacid, and/or co-factor.

A “conditioned medium” is prepared by culturing a first population ofcells in a medium, and then harvesting the medium. The conditionedmedium (along with anything secreted into the medium by the cells) maythen be used to support the growth of a second population of cells.Where a particular ingredient or factor is described as having beenadded to the medium, what is meant is that the factor (or a cell orparticle engineered to secrete the factor) has been mixed into themedium by deliberate manipulation.

A “defined minimal growth medium” is comprised of essential minerals,amino acids, vitamins and substrates for the culture of cells. A typicaldefined growth medium may comprise one or more inorganic salt, aminoacid, vitamin, sugar, buffer, indicator dye or colourant.

A “fresh medium” is a medium that has not been purposely conditioned byculturing with a different cell type before being used with the celltype it is ultimately designed to support. Otherwise, no limitations areintended as to its manner of preparation, storage, or use. It is addedfresh (by exchange or infusion) into the ultimate culture, where it maybe consumed or otherwise processed by the cell types that are present.

An “osteogenic medium” is composed of essential minerals, amino acids,vitamins and substrates for the culture of cells as well as factorsnecessary for differentiating cells into osteocytes. A typicalosteogenic medium may be composed of a defined growth mediumsupplemented with one or more serum protein, amino acid, non-essentialamino acid, and/or co-factor.

“Feeder cells” or “feeders” are terms used to describe cells of one typethat are co-cultured with cells of another type, to provide anenvironment in which the cells of the second type can grow. Certaintypes of pPS cells can be supported by primary mouse embryonicfibroblasts, immortalized mouse embryonic fibroblasts, or humanfibroblast-like cells differentiated from hES cell. pPS cell populationsare said to be “essentially free” of feeder cells if the cells have beengrown through at least one round after splitting in which fresh feedercells are not added to support the growth of pPS cells.

The term “embryoid bodies” is a term of art synonymous with “aggregatebodies”, referring to aggregates of differentiated and undifferentiatedcells that appear when pPS cells overgrow in monolayer cultures, or aremaintained in suspension cultures. The starting material for forming anembryoid body is a culture of undifferentiated pluripotent stem cells.Embryoid bodies are a mixture of different cell types, typically fromseveral germ layers, as well at least some pluripotent cellsdistinguishable by morphological criteria and cell markers detectable byimmunocytochemistry. Typically the number of pluripotent stem cells inthe embryoid body decreases over time as the number of differentiatedcells increases. Differentiation occurring in the context of an embryoidbody is essentially a random event, thus each embryoid body culturedunder identical conditions will typically not be identical in terms ofthe cellular composition. The term is distinguished from a constructculture used in the generation of chondrocyte lineage cells in that thestarting material for a construct culture is not a culture that isprimarily comprised of undifferentiated pluripotent stem cells, butrather is comprised of cells that have begun to differentiate away fromthe pluripotent state typically down the chondrocyte lineage pathway.Thus the starting material of a construct is a more developmentallyadvanced cell. Moreover construct cultures typically comprise one ormore factors that specifically direct the differentiation of the culturedown a desired pathway such as the chondrocytic pathway.

A “growth environment” is an environment in which cells of interest willproliferate, differentiate, or mature in vitro. Features of theenvironment include the medium in which the cells are cultured, anygrowth factors or differentiation-inducing factors that may be present,and a supporting structure (such as a substrate on a solid surface) ifpresent.

A cell is said to be “genetically altered”, “transfected”, or“genetically transformed” when a polynucleotide has been transferredinto the cell by any suitable means of artificial manipulation, or wherethe cell is a progeny of the originally altered cell that has inheritedthe polynucleotide. The polynucleotide will often comprise atranscribable sequence encoding a protein of interest, which enables thecell to express the protein at an elevated level. The genetic alterationis said to be “inheritable” if progeny of the altered cell have the samealteration.

Treat, treatment, treating, as used herein means any of the following:the reduction in severity of a disease or condition; the reduction inthe duration of a disease course; the amelioration of one or moresymptoms associated with a disease or condition; the provision ofbeneficial effects to a subject with a disease or condition, withoutnecessarily curing the disease or condition; the prophylaxis of one ormore symptoms associated with a disease or condition.

General Techniques

General methods in molecular genetics and genetic engineering aredescribed in the current editions of Molecular Cloning: A LaboratoryManual, (Sambrook et al., Cold Spring Harbor); Gene Transfer Vectors forMammalian Cells (Miller & Calos eds.); and Current Protocols inMolecular Biology (F. M. Ausubel et al. eds., Wiley & Sons). Cellbiology, protein chemistry, and antibody techniques can be found inCurrent Protocols in Protein Science (J. E. Colligan et al. eds., Wiley& Sons); Current Protocols in Cell Biology (J. S. Bonifacino et al.,Wiley & Sons) and Current Protocols in Immunology (J. E. Colligan et al.eds., Wiley & Sons.). Reagents, cloning vectors, and kits for geneticmanipulation referred to in this disclosure are available fromcommercial vendors such as BioRad, Stratagene, Invitrogen, ClonTech, andSigma-Aldrich Co.

Cell culture methods are described generally in the current edition ofCulture of Animal Cells: A Manual of Basic Technique (R. I. Freshneyed., Wiley & Sons); General Techniques of Cell Culture (M. A. Harrison &I. F. Rae, Cambridge Univ. Press), and Embryonic Stem Cells: Methods andProtocols (K. Turksen ed., Humana Press). Other references of interestinclude Culture Is Our Business (M. McLuhan, Ballantine Books, 1970);and Understanding Media (M. McLuhan, Signet, 1970). Tissue culturesupplies and reagents are available from commercial vendors such asGibco/BRL, Nalgene-Nunc International, Sigma Chemical Co., and ICNBiomedicals.

General aspects of the biology and pathology of cartilage, and the roleof chondrocytes in the maintenance of joints can be found in thefollowing reference textbooks: Mechanobiology: Cartilage andChondrocyte, by J. F. Stoltz ed., IOS Press 2000; Biological Regulationsof the Chondrocytes, by M. Adolphe ed., CRC Press 1992; Bone andCartilage Allografts, by G. E. Friedlaender et al. eds., Amer. Acad.Orthopaedic 1991; Joint Cartilage Degradation, by J. F. Woessner & D. S.Howell eds., Marcel Dekker 1992; Skeletal Tissue Mechanics, 2^(nd)edition by R. B. Martin et al., Springer Verlag 1998; Molecular andDevelopmental Biology of Cartilage, B. De Crombrugghe et al. eds., Ann.N.Y. Acad. Sci. Vol. 785: 1996; and Joint Structure and Function: AComprehensive Analysis, 3^(rd) edition by P. K. Levangie et al. eds, F ADavis, 2000.

Preparation of Chondrocytes and Chondrocyte Progenitor Cells

Methods of the present invention provide a system as defined herein forthe preparation of chondrocytes and chondrocyte progenitor or precursorcells. Cultures of hES cells are grown adherently, without forming anembryoid body to more than three-quarters complete confluency understandard hES culture conditions.

The media is then changed for a chondrogenic differentiation media asdescribed herein and cultured for an appropriate period, e.g., about 13to 15 days, suitably 14 days, washed and placed into de-differentiationmedia and cultured adherently without forming a construct culture for afurther period as appropriate e.g., 4 to 6 days, suitably 5 days,trypsinised and resuspended. At this point the cells are distinct fromisolated primary chondrocytes in terms of morphology and properties.Further culture of the cells in chondrogenic media for a further periodof 21 days enables chondrocytes to be produced. However, culture of thesame chondrocyte precursors in osteogenic media (comprisingDexamethasone, beta glycerol phosphate, ascorbic acid, sodium pryuvateand L-glutamine. A suitable base media may include commerciallyavailable Knockout™ D-MEM (Invitrogen) which may be supplied with serum)does not lead to the derivation of functional osteoblasts.

Suitable types of chondrogenic media include a media comprisingdexamethasone, insulin, transferrin, selenious acid, bovine serumalbumin, and linoleic acid, L-proline, ascorbic acid, sodium pyruvateand a TGF-β(for example TGF-β3). A suitable base media may includecommercially available media such as DMEM (Life Technologies). Thede-differentiation media may be DMEM supplemented with serum.

Suitable markers of chondrocyte differentiation include Collagen II andaggrecan, and suitable markers of pluripotency include Oct4, Tra-1-60and Nanog.

Sources of Stem Cells

Embryonic stem cells can be isolated from blastocysts of members of theprimate species (U.S. Pat. No. 5,843,780; Thomson et al., Proc. Natl.Acad. Sci. USA 92:7844, 1995). Human embryonic stem (hES) cells can beprepared from human blastocyst cells using primary mouse fibroblastfeeder cells, according to the techniques described by Thomson et al.(U.S. Pat. No. 6,200,806; Science 282:1145, 1998; Curr. Top. Dev. Biol.38:133, 1998) and Reubinoff et al., Nature Biotech. 18:399, 2000. hEScell lines can also be derived on human feeders (U.S. Pat. No.6,642,048), or in conditions entirely free of feeder cells (US2002/0081724) or Klimanskaya et al., Lancet, 365(9471):1636-41 (2005)).Equivalent cell types to hES cells include their pluripotentderivatives, such as primitive ectoderm-like (EPL) cells, as outlined inWO 01/51610. Embryonic stem cells may be chosen from embryonic stem celllines or may be obtained directly from primary embryonic tissue.

By no means does the practice of this invention require that a humanblastocyst be disaggregated in order to produce the hES or embryonicstem cells for practice of this invention. hES cells can be obtainedfrom established lines obtainable from public depositories (for example,the WiCell Research Institute, Madison Wis. USA, or the American TypeCulture Collection, Manassas Va., USA).

A number of embryonic stem cell lines have been established including,but not limited to, H1, H7, H9, H13 and H14 (Thompson et al.);hESBGN-01, hESBGN-02, hESBGN-03 (BresaGen, Inc., Athens, Ga.); HES-1,HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International, Inc.,Singapore); HSF-1, HSF-6 (University of California at San Francisco); I3, I 4, I 6 (Technion-Israel Institute of Technology, Haifa, Israel);UCSF-1 and UCSF-2 (Genbacev et al., Fertil. Steril. 83(5):1517-29,2005); lines HUES 1-17 (Cowan et al., NEJM 350(13):1353-56, 2004); andline ACT-14 (Klimanskaya et al., Lancet, 365(9471):1636-41, 2005).Established hES cell lines may be obtained from various sourcesincluding the UK Stem Cell Bank (National Institute for BiologicalStandards and Control, UK), the National Stem Cell Bank (University ofWisconsin-Madison, USA) or WiCell (Madison, Wis., USA).

Human Embryonic Germ (hEG) cells can be prepared from primordial germcells as described in Shamblott et al., Proc. Natl. Acad. Sci. U.S.A.95:13726, 1998 and U.S. Pat. No. 6,090,622. US 2003/0113910 reportspluripotent stem cells derived without the use of embryos or fetaltissue. It may also be possible to reprogram other progenitor cells intohES cells by using a factor that induces the pluripotent phenotype(Chambers et al., Cell 113:643, 2003; Mitsui et al., Cell 113:631,2003). Under appropriate conditions, any cell with appropriateproliferative and differentiation capacities can be used for thederivation of differentiated tissues for use according to thisinvention.

The propagation and maintenance of pPS cells such that the cells remainpluripotent has been described. pPS cells may be propagated andmaintained using either a feeder cell layer or feeder free conditions(see, e.g. U.S. Pat. No. 5,843,780; U.S. Pat. Nos. 6,090,622, 6,800,480,WO 01/51616; WO 03/020920; WO 06/017370; WO 07/002086; WO 09/099539; WO09/099555 and Xu et al. (2001) Nature Biotechnology 19:971,

Many suitable commercially available base media have been developed forculturing proliferative cell types and thus are suitable for culturingpPS cell such as hES cells. Exemplary are X-VIVO™ 10 expansion medium(Biowhittaker) and QBSF™-60 (Quality Biological Inc.). See also WO98/30679 (Life Technologies Inc.) and U.S. Pat. No. 5,405,772 (Amgen).The X-VIVO™ 10 formulation contains pharmaceutical grade human albumin,recombinant human insulin and pasteurized human transferrin. Exogenousgrowth factors, artificial stimulators of cellular proliferation orundefined supplements are not included in the X-VIVO™ 10 medium. Theyare also devoid of any protein-kinase C stimulators. QBSF™-60 is aserum-free formulation that contains recombinant or pasteurized humanproteins. Other potential alternatives are Ex-Cell VPRO™ medium made byJRH Biosciences, and HyQ CDM4™ made by Hyclone and mTESR™ from StemCellTechnologies.

The base medium may be supplemented with additives that promoteproliferation of the undifferentiated phenotype while inhibitingdifferentiation. Fibroblast growth factor at high concentration isespecially effective to promote hES cell proliferation withoutdifferentiation. Exemplary are basic FGF (FGF-2), and FGF-4, but othermembers of the family can also be used. Equivalent forms are specieshomologs, artificial analogs, antibodies to the respective FGF receptor,and other receptor activating molecules. It has been determined fromgene expression analysis that undifferentiated hES cells expressreceptors for acidic FGF (FGF-1). At a high concentration, FGF alone issufficient to promote growth of hES cells in an undifferentiated state.Concentrations of FGF effective for promoting undifferentiated hES cellgrowth on their own usually have a lower bound of about 20, 30, or 40ng/mL, with a practical upper bound of about 200, 500, or 1000 ng/mL.Concentrations of at least 60, 80, or 100 ng/mL bFGF are both reliableand cost effective. Equivalent concentrations of other forms and analogsof FGF can be determined empirically by weaning cultures from bFGF intothe proposed substitute, and monitoring the culture for differentiationaccording to the marker system described below.

pPS cells expanded by another culture method (or obtained from a primarysource) can be inoculated into a vessel adapted to keep the cells insuspension. The vessel walls may be typically inert or resistant toadherence of undifferentiated pPS cells. There may also be a means forpreventing the cells from settling out, such as a stirring mechanismlike a magnetically or mechanically driven stir bar or paddle, a shakingmechanism (typically attached to the vessel by the outside), or aninverting mechanism (i.e., a device that rotates the vessel so as tochange the direction of gravity upon the cells). The use of any suitableagitation means is contemplated

Vessels suitable for suspension culture for process development includethe usual range of commercially available spinner or shaker flasks.Fermenters suitable for commercial production are Celligen Plus™ (NewBrunswick Scientific Co.) and the Stirred-Tank Reactor™ (Applikon Inc.).Other suitable bioreactors include the Wave Bioreactor (GE Healthcare).These bioreactors can be continuously perfused with medium or used in afed-batch mode, and come in various sizes.

Additional details regarding the propagation of pPS cells in suspensionmay be found in WO 07/002086.

Optimization of the suspension culture system can be accomplished byempirical testing. Undifferentiated cells from a previous surface orsuspension culture can be passaged to the test condition, and culturedfor a week or more. The cells can be examined periodically forcharacteristics of hES cells, for example, using the marker systemdescribed in the next section. The cells can also be passaged back to awell-established culture system, and evaluated for classic morphologicalfeatures of undifferentiated cells as well as any of the markersassociated with pluripotent stem cells described herein.

The hES cells used according to the present invention are intendedultimately for differentiation into cells of the chondrocyte lineage.The appropriate test to use during culture may not be the marker profileof the undifferentiated culture, but rather the ability of the cells todifferentiate as required. The pluripotency of hES suspension culturescan be confirmed by sampling the cells, and either producing teratomasin SCID mice, or by staining EB-derived cells for markers representingall three germ layers. Markers for pluripotency include Oct4 and Nanog.

Alternatively or in addition, the suspension culture may containparticulate carriers that create surfaces within the suspension, butstill provide the benefits of culturing the cells in a three-dimensionalspace. The cells are cultured and passaged in the same way, except thatthe particles are retained in the vessel during medium exchange, andmore particles are added when the cells are split.

One type of microcarrier is solid spherical or semi-spherical particlesmade from glass, plastic, and dextran having a positive charge toaugment cell attachment (Cytodex), and so on. Another type isdisk-shaped culture plastic, such as the Fibra-cel Disks™ sold by NewBrunswick Scientific Co, Inc. A gram of these disks provide a surfacearea of 1200 cm². Another type of microcarrier is macroporous particlesof various pore sizes that permit the cells to reside in the interior aswell as the outside, to potentially enhance the protective effect. Inorder to recover the hES cells with minimal disruption, it is beneficialto use particles made of a material such as agarose that can easily bedissolved or dispersed by gentle mechanical or enzymatic action, therebyreleasing the cells for harvest or further culture. Solid carriers areoptionally coated with an hES cell friendly extracellular matrix, suchas laminin, Matrigel® or the like so that the attached cells have thesame microenvironment as cells plated onto a solid surface.

Characteristics of Undifferentiated hES Cells

Human ES cells have the characteristic morphological features ofundifferentiated stem cells. In the two dimensions of a standardmicroscopic image, hES cells have high nuclear/cytoplasmic ratios in theplane of the image, prominent nucleoli, and compact colony formationwith poorly discernable cell junctions. Cell lines can be karyotypedusing a standard G-banding technique (available at many clinicaldiagnostics labs that provide routine karyotyping services, such as theCytogenetics Lab at Oakland Calif.) and compared to published humankaryotypes. It is desirable to obtain cells that have a “normalkaryotype”, which means that the cells are euploid, wherein all humanchromosomes are present and are not noticeably altered.

hES cells can be characterized by expressed cell markers detectable byantibody (flow cytometry or immunocytochemistry) or by reversetranscriptase PCR. hES cells typically have antibody-detectable SSEA-3,SSEA-4, Tra-1-60, and Tra-1-81, but little SSEA-1, and have alkalinephosphatase activity. Panels of suitable markers detectable at the mRNAlevel are listed in US 2003/0224411. Exemplary are Cripto,gastrin-releasing peptide (GRP) receptor, podocalyxin-like protein(PODXL), human telomerase reverse transcriptase (hTERT), and the POUtranscription factor Oct 3/4.

As already described, an important feature of propagated hES cells is apotential to differentiate into cells of all three germ layers:endoderm, mesoderm, and ectoderm. Pluripotency of hES cells can beconfirmed by forming teratomas in SCID mice, and examining them forrepresentative tissues of all three germ layers. Alternatively,pluripotency can be determined by allowing hES cells to differentiatenon-specifically (for example, by forming embryoid bodies), and thendetermining the cell types represented in the culture byimmunocytochemistry.

Standard Methods for Differentiating Pluripotent Cells or ChondrocytePrecursor Cells into Chondrocytes

Chondrocytes can be obtained from DCCPC of this invention by culturing,differentiating, or reprogramming the chondrocyte precursor cells in aspecial growth environment that enriches for cells with the desiredphenotype (either by outgrowth of the desired cells, or by inhibition orkilling of other cell types). These methods are also applicable to manytypes of stem cells, including primate pluripotent stem (pPS) cells,including iPS cells, described herein.

When derived from an established line of pPS cells, the cell populationsand isolated DCCPC of this invention will have the same genome as theline from which they are derived. Reference to the cells having the samegenotype is not intended to imply that the cells cannot be geneticallymanipulated by the human hand (embodiments encompassing geneticallyaltered cells are described infra), or that very minor changes (e.g.,less than a fraction of a percent of the entire genome) might occurspontaneously (e.g. in the non-coding regions), but rather merely tosuggest that the act of differentiating the cells from pPS cells intocells of the chondrocyte lineage will not, by itself, result in analtered genotype. Typically the genetic identity between a parental(undifferentiated cell) and its differentiated progeny will be similarto the genetic identity found between identical twins. Typically the pPScell line and its DCCPC or chondrocyte progeny will share about 96%,about 97%, about 98%, about 99%, about 99.9% genetic identity.

The methods of the invention to prepare chondrocyte precursors cells donot require the formation of an embryoid body.

In certain embodiments the methods of the invention to prepare maturechondrocytes and/or cells expressing collagen II do not require theformation of a construct. In other embodiments the formation of aconstruct comprising chondrocyte progenitor cells may facilitatedifferentiation of the DCCPC into mature chondrocytes and/or cellsexpressing collagen II.

In order to direct the chondrocyte precursor cell culture towards thechondrocyte pathway, precursor cells that have been prepared asdescribed above can be cultured in a cocktail of chondrocytedifferentiation factors. Alone or in combination, each of the factorsmay direct cells to differentiate down the chondrocyte pathway, causeoutgrowth of cells with a chondrocyte phenotype, inhibit growth of othercell types, or enrich for chondrocytes in another fashion: it is notnecessary to understand the mechanism resulting in chondrocytes beingenriched in order to practice the invention.

Components of the chondrocyte differentiation mix may includetransforming growth factors (especially TGFβ1, TGFβ2 and TGFβ3),fibroblast growth factors (especially basic fibroblast growth factor,FGF-2), growth and differentiation factors (especially GDF-5, GDF-6 andGDF-7), bone morphogenic proteins (especially BMP-2, BMP-4, BMP-5, BMP-6and BMP-7), hedgehog proteins (especially Indian hedgehog, IHH),L-ascorbic acid, and parathyroid hormone-related protein (PTHrP). Gibcois a preferred source of basic FGF. Most of the other compounds areavailable from R&D Systems, Minneapolis Minn.

Other ligands or antibodies that bind the same receptors can beconsidered equivalents to any of the receptor ligands referred to inthis disclosure.

Transforming growth factors beta (TGFβ) regulate various aspects ofembryonic development and are expressed in the environment ofsympathoadrenal progenitor cells (Wall et al., Curr. Opin. Genet. Dev.4:517, 1994). In some systems, TGFβ regulates expression of parathyroidhormone-related protein (PTHrP) (Pateder et al., J. Cell Physiol.188:343, 2001). BMPs and growth and differentiation factors (GDFs) arebelieved to play a central role during skeletogenesis, including jointformation (Francis-West et al., Cell Tissue Res 1296:111, 1999). Indianhedgehog (IHH) is an essential component of mechanotransduction complexto stimulate chondrocyte proliferation (Wu et al., J. Biol. Chem.276:35290, 2001). During endochondral ossification, two secretedsignals, IHH and PTHrP are believed to form a negative feedback loopregulating the onset of hypertrophic differentiation of chondrocytes.Bone morphogenetic proteins (BMP) are thought to be mediators ofsignalling pathways for the patterning of skeletal elements andmechanisms for the induction of cartilage and bone formation. (Hoffmannet al., Crit. Rev. Eukaryot. Gene Expr. 11:23, 2001). BMPs have beenimplicated as potential interactors of the IHH/PTHrP feedback loop(Minina et al., Development 128:4523, 2001). BMPs may very well interactwith IHH and PTHrP to coordinate chondrocyte proliferation anddifferentiation.

Phenotypic Markers of Chondrocytes

Type II collagen and aggrecan can be used as specific markers for cellsthat model articular cartilage. Cultures can be screened for the absenceof elastin and Type I collagen, markers of elastic cartilage andfibrocartilage, respectively. Cultures may also be screened for theabsence of Type X collagen and osteocalcin, markers of hypertrophiccartilage and bone, respectively, which could indicate a transientchondrocyte phenotype generated during the progression of endochondralbone formation. A table of commercially available antibodies for thesehuman markers is shown below (Table 2).

Tissue-specific markers can be detected using any suitable immunologicaltechnique, such as flow immunocytochemistry for cell-surface markers, orimmunohistochemistry (for example, of fixed cells or tissue sections)for intracellular or cell-surface markers. A detailed method for flowcytometry analysis is provided in Gallacher et al., Blood 96:1740, 2000.Expression of a cell-surface antigen is defined as positive if asignificantly detectable amount of antibody will bind to the antigen ina standard immunocytochemistry or flow cytometry assay, optionally afterfixation of the cells, and optionally using a labelled secondaryantibody or other conjugate to amplify labelling. A significantlydetectable amount of antibody may for example be an amount in excesswhen compared to an isotype antibody control to an irrelevant epitope.Possible sources of specific antibody are shown in Table 2.

TABLE 2 Commercial Sources of Antibody to Connective Tissue MarkersAntibody Source   Type I collagen Chemicon, cat. # AB758 Type IIcollagen Chemicon, cat # AB761 Type X collagen RDI, cat # RDI-COLL10abrElastin Chemicon, cat # AB2043 Osteocalcin Biomed. Tech. Inc., cat #BT593 Aggrecan BioTrend, cat # 0195-8050

The expression of tissue-specific gene products can also be detected atthe mRNA level by Northern blot analysis, dot-blot hybridizationanalysis, or by reverse transcriptase initiated polymerase chainreaction (RT-PCR) using sequence-specific primers in standardamplification methods. See U.S. Pat. No. 5,843,780 for further details.Real time PCR may also be performed using commercially available systemssuch as TaqMan® (Applied Biosystems). Sequence data for particularmarkers listed in this disclosure can be obtained from public databasessuch as GenBank.

To facilitate engraftment, it is beneficial to maximize the proportionof cells in the population that have the characteristics of chondrocytesor their precursors, such as DCCPC, by refining the mixture ofdifferentiation factors, culture conditions, and timing and followingthese markers. Populations in which at least 5% of the cells synthesizeeither Type II collagen or aggrecan, or both, may well be suitable.Populations enriched to the point where at least 25% of the cellssynthesize either Type II collagen or aggrecan would be more efficaciousin a number of contexts.

For therapeutic applications relating to cartilage regeneration, it maybe desirable to minimize the ability of the cell population to formelastic cartilage, fibrocartilage, hypertrophic cartilage and bone. Thismeans that the proportion of cells synthesizing Type I collagen, Type Xcollagen, or osteocalcin (alone or in combination) is as low aspossible, preferably below ⅕^(th) of the cells staining positive forType II collagen or aggrecan, or less than 1%. Also desirable arepopulations with a low residual proportion of undifferentiated pPScells. Preferred populations are less than 1%, or 0.2% SSEA-4 positive(+ve), Oct-4 positive (+ve), or positive for expression of endogenoustelomerase reverse transcriptase. Any depletion technology known in theart may be used eliminate unwanted cell populations. For examplemagnetic beads conjugated with antibodies specific to an extra-cellularmarker may be used.

Animal Model Experiments

Of considerable interest for development of chondrocytes for clinicalapplication is the ability of cell populations to model cartilage andrestore joint function in a host. Reconstitution of chondrocyte functioncan be tested using several well-established animal models.

Pilot experiments can be conducted using a model in which 6 mm holes areput in the external ear of the rabbit, leaving the adherent skin intact.Matrixes seeded with chondrocytes are then implanted, and the animalsare monitored for hole closure (ten Koppel et al., Biomaterials 22:1407,2001).

Alternatively, full thickness defects can be created in the weightbearing surface of the medial femoral chondyle of femora in rabbits(Grigolo et al., Biomaterials 22:2417, 2001). The full-thickness defectallows blood to seep into the site from the marrow cavity, creating aclot that contains endogenous stem cells.

Alternatively, partial thickness defects that do not puncture thesubcutaneous bone can be created Nehrer et al., (Biomaterials19:2313-2328, 1998) (Hunziker et al., Clin. Orthop. 391 Supp:S171,2001). Partial thickness defects more accurately model acute cartilagedefects due to trauma. In this model the spontaneous healing componentof innate cartilage repair is reduced and endogenous stem cellscontribute little to cartilage regrowth.

The wounds are repaired using chondrocytes seeded on a biomaterial,injected as single cells or used as a multicellular aggregate from theconstruct culture. A biological membrane such as a piece of theperiosteum or facia may be used to hold the implant in place.Alternatively synthetic matrices such as vicryl or polydioxanone meshesmay be used. Matrix-cell implants can also be held in place withsurgical dart.

Rather than the medial chondyle, the work can be done with a defectcreated in the trochlear groove. This is a non-weight-bearing site, andimplants are not dislodged as easily as from the medial chondyle.

Histologic samples from the treatment site are examined 1-6 months aftersurgery for population with the implant cells and cartilage deposits.This includes immunostaining for Type II collagen and aggrecan.Important morphological characteristics of the implant include cartilagethickness, smooth articular surface, intact or reconstituted cement lineand integration of the implant and endogenous cartilage at the bordersof the defect. Long-term stability of the implant can be verified at the12 month point.

As another option, osteoarthritis can be modelled by the injection ofestradiol into the knee joint of ovarectomized rabbits, causing loss ofcondyle surface congruity that resemble the defects observed inosteoarthritis in humans (Tsai et al., Clin Orthoop. 291:295, 1993).Alternatively joint destabilisation via transaction of supportiveligaments may be used to model osteoarthritis (Glasson S.,OsteoArthritis and Cartilage (2007) 15, 1061-1069).

Other mammals may be used as an animal model. Suitable mammals includerodents such as rats and mice, ungulates such as pigs, sheep cows andhorses, felines, canines and non-human primates.

Animal models described above may also be used to investigate acceptanceor rejection of implanted cells. Accordingly evidence of immunerejection may be investigated. Evidence of immune rejection may includeleukocyte infiltration into the implant site, evidence of inflammationsuch as the presence of pro-inflammatory cytokines. Loss of implantedcells may also suggest immune rejection. Other signs of immune rejectionmay include lymphocyte proliferation and stimulation of interferon gammaproduction.

Genetic Modification of Differentiated Cells

Certain chondrocyte precursor cell populations of this invention, suchas DCCPC have a substantial proliferation capacity. If desired, thereplication capacity can be further enhanced by increasing the level oftelomerase reverse transcriptase (TERT) in the cell, either byincreasing transcription from the endogenous gene, or introducing atransgene. Particularly suitable is the catalytic component of humantelomerase (hTERT), provided in International Patent Application WO98/14592. Transfection and expression of telomerase in human cells isdescribed in Bodnar et al., Science 279:349, 1998 and Jiang et al., Nat.Genet. 21:111, 1999. Genetically altered cells can be assessed for hTERTexpression by RT-PCR, telomerase activity (TRAP assay),immunocytochemical staining for hTERT, or replicative capacity,according to standard methods. Other methods of immortalizing cells arealso contemplated, such as transforming the cells with DNA encoding myc,the SV40 large T antigen, or MOT-2 (U.S. Pat. No. 5,869,243, WO 97/32972and WO 01/23555).

If desired, the cells of this invention can be prepared or furthertreated to remove undifferentiated cells in vitro, or to safeguardagainst revertants in vivo. One way of depleting undifferentiated stemcells from the population is to transfect the population with a vectorin which an effector gene under control of a promoter that causespreferential expression in undifferentiated cells—such as the TERTpromoter or the OCT-4 promoter. The effector gene may be a reporter toguide cell sorting, such as green fluorescent protein. The effector maybe directly lytic to the cell, encoding, for example, a toxin, or amediator of apoptosis, such as caspase (Shinoura et al., Cancer GeneTher. 7:739, 2000). The effector gene may have the effect of renderingthe cell susceptible to toxic effects of an external agent, such as anantibody or a prodrug. Exemplary is a herpes simplex thymidine kinase(tk) gene, which causes cells in which it is expressed to be susceptibleto ganciclovir (WO 02/042445). Alternatively, the effector can causecell surface expression of a foreign determinant that makes any cellsthat revert to an undifferentiated phenotype susceptible to naturallyoccurring antibody in vivo (WO 02/042445). Other ways of eliminatingunwanted pluripotent cells include using immuno-precipitating reagentssuch as a bead conjugated with an antibody to a cell surface proteinexpressed on a pluripotent stem cell.

Uses of Propagated Chondrocyte Cells

This invention provides a method by which large numbers of chondrocyteor DCCPC can be produced on a commercial scale from pPS cells, inparticular hES cells. The chondrocyte or DCCPC are useful for a numberof research and commercial purposes.

Therapeutic Uses

This invention also provides for the use of DCCPC and their derivativesto treat conditions leading to impairment of joint mobility or defectsor depletions relating to the in vivo functional capability ofendogenous cartilage. Suitable subjects include any mammal such as arat, a mouse, a rabbit, a pig, a cow, a horse, a sheep, a cat, a dog, anon-human primate such as a chimpanzee or a macaque, and a human.

Included is damage caused by percussive trauma, and sports injuries. Thecells of the invention can also be considered for treatment ofdegenerative conditions, such as osteoarthritis and rheumatoid arthritisto restore lost function, providing that the primary pathology causingthe degeneration is sufficiently well controlled. Also contemplated isthe use of the cells of this invention for cosmetic surgery, includingbut not limited to ear, spine and nasal surgery (i.e. of the proboscis).See Aesthetic Reconstruction of the Nose, by G. C. Burget & F. J.Menick, Mosby Year Book 1994; The Nose, by Nikolay Gogol et al., DavidR. Godine publisher, 1993; and Yoo et al., J. Urol. 162:1119, 1999.

DCCPC and/or chondrocytes made according to this invention can beprepared for administration in a cell suspension (using trypsin orcollagenase, if necessary), using a physiologically compatibleexcipient: for example, an isotonic medium containing 1.25 mL gentomycinsulfate (70 μmol/L), 2.0 mL amphotericin (2.2 μmol/L), 7.5 mL L-ascorbicacid (300 μmol/L), 25 mL blood serum or its equivalent (to 10% vol/vol)in 300 mL. During the cell transplantation procedure, the damagedcartilage can be covered with a cap secured by mechanical or adhesiveretention means, such as a biocompatible fibrin glue. The cultured cellsin transplant excipient (about 0.6 mL containing about 10×10⁶chondrocytes) may then be injected under the covering cap using a ˜23gauge needle.

Alternatively, the DCCPC and/or chondrocytes can be prepared by growingthe cells on a matrix formed, for example, using a collagen membrane(commercially available collagen matrix pads are available from Ed.Geistlich Sohne, Switzerland). A few days before transplant, the growthmedia is exchanged for a transplant excipient. During surgery, thecell-loaded matrix is glued into the area of damaged cartilage using abiocompatible adhesive. The covering cap or matrix remains in place fora period of time sufficient to allow for cartilage repair, and is thenabsorbed or resorbed by the body, for example, within two to threemonths from implantation. Further elaboration of the design and use ofresorbable caps and matrices can be found in International patentpublication WO 01/08610.

Patient selection, mode of administration, and choice of supportstructures and surgical options is within the skill of the managingclinician.

For purposes of commercial distribution, chondrocytes of this inventionare typically supplied in the form of a pharmaceutical composition,comprising an isotonic excipient prepared under sufficiently sterileconditions for human administration. For general principles in medicinalformulation of cell compositions, the reader is referred to CellTherapy: Stem Cell Transplantation, Gene Therapy, and CellularImmunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge UniversityPress, 1996. The composition may also contain a matrix for keeping thechondrocytes in place during the first few months following therapy.Absorbable biomaterials save the necessity of subsequent surgicalremoval. Besides the collagen matrix pads described in WO 01/086101,other possible matrixes include bioresorbable polymer fleece matrices(Rudert et al., Cells Tissues Organs 167:95, 2000); hyaluronanderivatives (Grigolo et al., Biomaterials 22:2417, 2001); sponge madefrom poly(L-lactide-epsilon-caprolactone) (Honda et al., J. OralMaxillofac. Surg. 58:767, 2000), and collagen-fibrin matrices (Clin.Exp. Rheumatol. 18:13, 2000).

Screening Uses

The chondrocytes or DCCPC can be used to screen for factors (such assmall molecule drugs, peptides, polynucleotides, and the like) orconditions (such as culture conditions or manipulation) that affect thecharacteristics of chondrocytes or DCCPC in culture. Such cultures canalso be used for the testing of pharmaceutical compounds in drugresearch. Assessment of the activity of candidate pharmaceuticalcompounds generally involves combining the differentiated cells of thisinvention, such as chondrocytes or DCCPC with the candidate compound,determining any resulting change, and then correlating the effect of thecompound with the observed change. Comparisons can be made to anequivalent culture that has not been treated with the factor orcompound. Cytotoxicity or metabolic effects can be determined by cellviability, morphology, the expression or release of certain markers,receptors or enzymes, DNA synthesis or repair, and so on. The cellsprepared according to the present invention can be used for drugscreening, preparing pharmaceutical compositions, research, and manyother similar purposes.

In one example, the DCCPC can be used to screen factors that promotematuration into chondrocytes, or promote proliferation and maintenanceof chondrocytes in long-term culture. For example, candidate maturationfactors or growth factors can be tested by adding them to cells indifferent wells, and then determining any phenotypic change that results(such as expression of collagen II and/or aggrecan), according todesirable criteria for further culture and use of the cells. Comparisonscan be made to an equivalent culture that has not been treated with thefactor. This can lead to improved derivation and culture methods notonly for pPS derived chondrocytes, but for chondrocytes and theirprogenitors isolated from cartilage.

Another example is the use of DCCPC to measure the effect of moleculescapable promoting chondrocyte survival under conditions of stress. Forexample those associated with trauma through injury, surgery orosteoarthritis.

Another example is the use of chondrocyte precursors to measure theeffect of small molecule drugs that have the potential to affectchondrocyte activity in their role of shaping or remodelling cartilage.To this end, the cells can be combined with test compounds in vitro, andthe effect of the compound on gene expression or protein synthesis canbe determined. The screening can also be done in vivo by measuring theeffect of the compound on the behaviour of the cells in an animal model.Untreated cells or animals may be used for comparison.

Other screening methods of this invention relate to the testing ofpharmaceutical compounds for a potential effect on chondrocyte growth,development, or toxicity. This type of screening is appropriate not onlywhen the compound is designed to have a pharmacological effect onchondrocytes themselves, but also to test for chondrocyte-relatedside-effects of compounds designed for a primary pharmacological effectelsewhere.

Assessment of the activity of candidate pharmaceutical compoundsgenerally involves combining the differentiated cells of this inventionwith the candidate compound, either alone or in combination with otherdrugs (“In vitro Methods in Pharmaceutical Research”, Academic Press,1997; and U.S. Pat. No. 5,030,015). The investigator determines anychange in the morphology, marker phenotype, or functional activity ofthe cells that is attributable to the compound (compared with untreatedcells or cells treated with an inert compound), and then correlates theeffect of the compound with the observed change. Untreated cells may beused for comparison.

Cytotoxicity can be determined in the first instance by the effect oncell viability, survival, morphology, and the expression of certainmarkers and receptors. Effects of a drug on chromosomal DNA can bedetermined by measuring DNA synthesis or repair. [³H]-thymidine or BrdUincorporation, especially at unscheduled times in the cell cycle, orabove the level required for cell replication, is consistent with a drugeffect. Unwanted effects can also include unusual rates of sisterchromatid exchange, determined by metaphase spread (A. Vickers, pp375-410 in “In vitro Methods in Pharmaceutical Research,” AcademicPress, 1997).

Commercial Distribution

Components of the chondrocyte differentiation culture system of thisinvention may be prepared together in various useful combinations, suchas two or more of the following:

-   -   media suitable for culturing DCCPC and/or chondrocytes in        suspension or adherently    -   extracellular matrix components or thickeners present in or to        be added to the medium or coated onto a suitable cell growth        surface    -   microcarriers present in or to be added to the medium    -   vessels adapted for suspension culture or adherent culture    -   the chondrocytes or DCCPC themselves, either growing in a        culture system, or stored in another form, but intended for use        in a culture system    -   pPS cells in culture or stored in a suitable excipient or buffer    -   one or more cytokines, growth factors, morphogens or the like        suitable for promoting the growth, differentiation and/or        maturation of pPS cells and/or chondrocyte precursor cells into        mature chondrocytes

The products and product combinations may be packaged in suitablecontainers, optionally in kit form, and may be accompanied by writteninformation on the use of the materials according to this invention—suchas maintaining or expanding chondrocyte cells. The written informationmay take the form of a label on the container or the kit, or a productinsert packaged with the container and distributed together. Equivalentforms are descriptions, instructions, or explanations written in hardcopy or in electronic form available to the user or the intended user asreference or resource material associated with the commerciallydistributed product.

This invention may also include sets of cells and other components thatexist at any time during their manufacture, distribution, or use. Thecell sets can comprise any combination of two or more cell populationsdescribed in this disclosure, exemplified but not limited to a type ofdifferentiated pPS-derived chondrocyte precursor cell prepared by amethod of the invention, such as DCCPC in combination withundifferentiated pPS cells or other differentiated cell types (e.g.fully differentiated chondrocytes), sometimes sharing the same genome.Each cell type in the set may be packaged together, or in separatecontainers. The products of this invention are optionally packaged in asuitable container or kit with written instructions for a desiredpurpose, such as the treatment of a cartilage defect, the reconstructionof joint cartilage or cosmetic surgery.

The present invention has shown that ES cells differentiated for 14 daysin chondrogenic media are able to revert to a dedifferentiated state(DCCPC) if the differentiation factors present in the media are removed.Cells de-differentiated are valuable for a number of reasons: 1) theinitial differentiation step using the chondrogenic protocol describedherein is effective at removing or differentiating pluripotent cells,thus limiting their ability to form teratomas; 2) de-differentiationallows the cells to proliferate and provide a second bulking up stage;and 3) the de-differentiated cells from primary chondrocytes have beenshown to be plastic adherent and will passage as single cells (unlikehESCs). The de-differentiated cells are stable and can be subsequentlydifferentiated into collagen II expressing cells and/or maturechondrocytes either under adherent conditions or when grown insuspension as a construct. These features are highly beneficial to largescale production of a cell therapy.

Additional Embodiments of the Invention

1. Cell Cultures and Cell Populations

In some embodiments the invention provides a cell culture comprising apopulation of cells wherein the population of cells are the in vitroprogeny of pPS cells and wherein the population of cells expressCBFA1/RunX2, and are Ki67 negative and negative for pluripotency markersTra 1-60, Oct 4 and nanog. The cell population may also be negative forpluripotency markers SSEA 3 and SSEA 4.

In certain embodiments the cell culture never comprises an embryoidbody. In some embodiments the cell culture comprises only adherentcells. In some embodiments the culture does not comprise a construct. Inother embodiments the cell culture may comprise a construct of cells.

In some embodiments of the invention the cell culture comprises cellswherein more than 10%, more than 20%, more than 30%, more than 40%, morethan 50%, more than 60%, more than 70%, more than 80% of the cells inthe cell culture express CBFA1/RunX2. In some embodiments of theinvention 85% of the cells in the population express CBFA1/RunX2.

In some embodiments of the invention the cell culture comprises cellswherein less than 20%, less than 10%, less than 5%, less than 2%, lessthan 1% of the cells in the cell culture express one or more of thefollowing markers: Tra 1-60, Oct 4, nanog, SSEA4, SSEA 3 and Ki67. Inother embodiments of the invention none of the cells in the cell cultureare detectible for at least one of the following markers: Tra 1-60, Oct4, nanog, SSEA4, SSEA 3 and Ki67.

In some embodiments the cell culture is provided on a surface such thatthe cells are in direct contact with a plastic surface e.g. a plastictissue culture dish. In other embodiments the culture is provided onsubstrate that coats a tissue culture surface. In some embodiments thesubstrate may be comprised of one or more extra cellular matrixcomponents. In some embodiments the substrate may be comprised oflaminin. In some embodiments the substrate may comprise the extract of amurine sarcoma cell, e.g. Matrigel®. In some embodiments the substrateis not collagen. In some embodiments the substrate is not gelatin.

In some embodiments the cell culture comprises a nutrient media. Thenutrient media may be comprised of serum such as fetal calf serum or thelike. In some embodiments the media may be comprised of about 5-20%serum. In some embodiments the media is comprised of 10% serum. In someembodiments the nutrient media is a commercially available media such asDMEM. In other embodiments the cell culture comprises adedifferentiation media, e.g. a media that is 10% FBS such as DMEM(Invitrogen). In some embodiments the nutrient media does not compriseexogenously added TGFβ1, FGF2, and PDGFbb beyond what is found in amedia comprising 10% FCS.

2. Systems for Producing Chondrocyte Lineage Cells

In certain embodiments the invention provides a system for producingchondrocyte lineage cells. Chondrocyte lineage cells may include maturechondrocytes, DCCPC, and/or cells expressing one or more of thefollowing: collagen II, aggrecan, glycosamino-glycan.

The system for producing chondrocyte lineage cells may comprise 1) afirst population of cells comprising pPS cells and 2) a secondpopulation of cells comprising DCCPC wherein the DCCPC are the in vitroprogeny of a portion of the pPS cells.

In further embodiments the invention provides a system for producingchondrocyte lineage cells comprising 1) a first population of cellscomprising pPS cells and 2) a second population of cells comprisingcells that express CBFA1/RunX2 and do not express Ki67, wherein thecells expressing CBFA1/RunX2 without expressing the proliferation markerKi67 are the in vitro progeny of a portion of the pPS cells.

Because pPS cells may be established as cell lines and thus growncontinuously in culture while maintaining their pluripotent state theycan produce chondrocyte lineage cells in virtually unlimited supply. Thechondrocyte lineage cells produced will be essentially geneticallyidentical to the parent pPS cell line. Thus the system provides for acontinual unlimited source of chondrocyte lineage cells that areessentially genetically identical to one another.

In some embodiments the system is never comprised of an embryoid body.In some embodiments the system is never comprised of a construct. Insome embodiments the cells are maintained adherently throughout (exceptfor the time required for passaging e.g. trypsinization or the like). Insome embodiments of the invention one or both populations of cellpopulations comprising the system may be provided on a plastic surfacesuch as the surface of a cell culture article. In other embodiments ofthe invention one or both of the cell populations comprising the systemare provided on a substrate that coats a tissue culture surface. In someembodiments the substrate may be comprised of one or more extra cellularmatrix components. In some embodiments the substrate may be comprised oflaminin. In some embodiments the substrate may comprise the extract of amurine sarcoma cell, e.g. Matrigel®. In some embodiments the substrateis not collagen. In some embodiments the substrate is not gelatin. Inother embodiments the second population of cells may be provided as aconstruct. The construct may be provided as a non-adherent cellpopulation, e.g. a cell population that is not attached to either aplastic surface or an exogenously provided substrate such as a cellmatrix. The cells of the construct may be attached to other cells withinthe construct.

In some embodiments of the invention the system may further comprise oneor more nutrient media. In certain embodiments of the invention thefirst population of cells is provided in a media comprising one or moreof the following: linoleic acid and bovine serum albumin. In someembodiments the media may further comprise one or more of the following:dexamethasone, insulin, transferrin, selenium, ascorbic acid, sodiumpyruvate, and transforming growth factor β (TGFβ), e.g. TGFβ3. Incertain embodiments the media does not comprise an exogenously addedbone morphogenic protein. In certain embodiments the media does notcomprise a serum replacement such as knock out serum replacement.

A suitable media concentration of linoleic acid may range from about 2mg/ml to about 10 mg/ml; from about 3 mg/ml to about 7 mg/ml; from about4 mg/ml to about 6 mg/ml. In one embodiment of the invention the mediais comprised of about 5.35 mg/ml of linoleic acid. A suitable mediaconcentration of bovine serum albumin may range from about 0.5 mg/ml toabout 5 mg/ml; from about 0.8 mg/ml to about 3 mg/ml; from about 1 mg/mlto about 2 mg/ml. In one embodiment of the invention the media iscomprised of about 1.25 mg/ml of bovine serum albumin. A suitable mediaconcentration of dexamethasone may range from about 10⁻⁸M to about 10⁻⁶;from about 10⁻⁷ M to about 10⁻⁶M; from about 10⁻⁸M to about 10⁻⁷ M. Inone embodiment of the invention, the media is comprised of about 10⁻⁷ Mdexamethasone. A suitable media concentration of insulin may range fromabout 3 ng/ml to about 20 ng/ml; from about 5 ng/ml to about 15 ng/ml;from about 6 ng/ml to about 9 ng/ml. In one embodiment of the inventionthe media is comprised of about 6.25 ng/ml insulin. A suitable mediaconcentration of transferrin may range from about 3 ng/ml to about 20ng/ml; from about 5 ng/ml to about 15 ng/ml; from about 6 ng/ml to about9 ng/ml. In one embodiment of the invention the media is comprised ofabout 6.25 ng/ml transferrin. A suitable media concentration ofselenious acid may range from about 3 ng/ml to about 20 ng/ml; fromabout 5 ng/ml to about 15 ng/ml; from about 6 ng/ml to about 9 ng/ml. Inone embodiment of the invention the media is comprised of about 6.25ng/ml selenious acid. A suitable media concentration of proline mayrange from about 20 μg/ml to about to about 80 μg/ml; from about 30μg/ml to about 70 ug/ml; from about 40 μg/ml to about 60 μg/ml. In oneembodiment of the invention the is comprised of about 40 μg/ml ofproline. A suitable media concentration of ascorbic acid may range fromabout 30 μg/ml to about 90 μg/ml; from about 40 μg/ml to about 80 μg/ml;from about 45 μg/ml to about 60 μg/ml. In one embodiment of theinvention the media is comprised of about 50 μg/ml of ascorbic acid. Asuitable media concentration of TGFβ may range from about 1 ng/ml toabout 30 ng/ml; from about 5 ng/ml to about 20 ng/ml; from about 8 ng/mlto about 15 ng/ml. In one embodiment of the invention the media iscomprised of about 10 ng/ml of TGFβ.

In some embodiments of the invention the second population of cells isprovided in a nutrient media such as a commercially available media,e.g. DMEM (Invitrogen). In some embodiments the nutrient media does notcomprise exogenously added TGFβ3, FGF2, and PDGFbb beyond what is foundin a media comprising 10% FCS.

In some embodiments the second population of cells is provided in anutrient media comprising serum. The nutrient media may be comprised ofserum such as fetal calf serum or the like. In some embodiments themedia may be comprised of about 5-20% serum. In some embodiments themedia is comprised of 10% serum. In some embodiments the nutrient mediais a commercially available media such as DMEM. In other embodiments thecell culture comprises a dedifferentiation media, e.g. a media that is10% FBS such as DMEM (Invitrogen).

In some embodiments of the invention the second population of cells maybe transferred from a nutrient media comprising 10% serum to the mediaused for culturing the first population of cells.

3. Methods for Producing Dedifferentiated Committed ChondrocyteProgenitor Cells (DCCPCs)

In certain embodiments the invention provides a method of producing(DCCPCs) comprising 1) culturing pPS cells in a chondrogenic media and2) removing the chondrogenic media and substituting a dedifferentiationmedia in its place.

In other embodiments the invention provides a method of producing a cellthat expresses CBFA1/RunX2 comprising) culturing pPS cells in achondrogenic media and 2) removing the chondrogenic media andsubstituting a dedifferentiation media in its place. The cellsexpressing CBFA1/RunX2 may be negative for Ki67. The cells expressingCBFA1/RunX2 may be negative for one or more of the following markersOct4, nanog, SSEA4, SSEA3, TRA-1-60. The cells expressing CBFA1/RunX2may have a fibroblast like morphology.

In certain embodiments of the invention the chondrogenic media comprisesone or more of: linoleic acid and bovine serum albumin. The media mayfurther comprise one or more of the following: dexamethasone, insulin,transferrin, selenium, ascorbic acid, sodium pyruvate, and transforminggrowth factor β (TGFβ), e.g. TGFβ3. In certain embodiments the mediadoes not comprise an exogenously added bone morphogenic protein. Incertain embodiments the media does not comprise a serum replacement suchas knock out serum replacement.

A suitable media concentration of linoleic acid may range from about 2mg/ml to about 10 mg/ml; from about 3 mg/ml to about 7 mg/ml; from about4 mg/ml to about 6 mg/ml. In one embodiment of the invention the mediais comprised of about 5.35 mg/ml of linoleic acid. A suitable mediaconcentration of bovine serum albumin may range from about 0.5 mg/ml toabout 5 mg/ml; from about 0.8 mg/ml to about 3 mg/ml; from about 1 mg/mlto about 2 mg/ml. In one embodiment of the invention the media iscomprised of about 1.25 mg/ml of bovine serum albumin. A suitable mediaconcentration of dexamethasone may range from about 10⁻⁸M to about 10⁻⁶;from about 10⁻⁷ M to about 10⁻⁶M; from about 10⁻⁸ M to about 10⁻⁷ M. Inone embodiment of the invention the media is comprised of about 10⁻⁷ Mdexamethasone. A suitable media concentration of insulin may range fromabout 3 ng/ml to about 20 ng/ml; from about 5 ng/ml to about 15 ng/ml;from about 6 ng/ml to about 9 ng/ml. In one embodiment of the inventionthe media is comprised of about 6.25 ng/ml insulin. A suitable mediaconcentration of transferrin may range from about 3 ng/ml to about 20ng/ml; from about 5 ng/ml to about 15 ng/ml; from about 6 ng/ml to about9 ng/ml. In one embodiment of the invention the media is comprised ofabout 6.25 ng/ml transferrin. A suitable media concentration ofselenious acid may range from about 3 ng/ml to about 20 ng/ml; fromabout 5 ng/ml to about 15 ng/ml; from about 6 ng/ml to about 9 ng/ml. Inone embodiment of the invention the media is comprised of about 6.25ng/ml selenious acid. A suitable media concentration of proline mayrange from about 20 μg/ml to about to about 80 μg/ml; from about 30μg/ml to about 70 μg/ml; from about 40 μg/ml to about 60 μg/ml. In oneembodiment of the invention the is comprised of about 40 μg/ml ofproline. A suitable media concentration of ascorbic acid may range fromabout 30 μg/ml to about 90 μg/ml; from about 40 μg/ml to about 80 μg/ml;from about 45 μg/ml to about 60 μg/ml. In one embodiment of theinvention the media is comprised of about 50 μg/ml of ascorbic acid. Asuitable media concentration of TGFβ may range from about 1 ng/ml toabout 30 ng/ml; from about 5 ng/ml to about 20 ng/ml; from about 8 ng/mlto about 15 ng/ml. In one embodiment of the invention the media iscomprised of about 10 ng/ml of TGFβ.

In some embodiments of the invention the dedifferentiation mediacomprises a commercially available media, e.g. DMEM (Invitrogen). Themedia may be comprised of serum such as fetal calf serum (FCS). Incertain embodiments the media is about 10% FCS. In some embodiments themedia does not comprise exogenously added TGFβ1, FGF2, and PDGFbb beyondwhat is found in a media comprising 10% FCS.

In some embodiments of the invention the method of producing DCCPCscomprises culturing the pPS cells adherently. In some embodiments of theinvention the pPS cells never form an embryoid body. In certainembodiments of the invention the method may further comprise removingthe cells from the adherent surface after they have been cultured in thechondrogenic media, pelleting the cells, e.g. by centrifugation,resuspending the cells in the dedifferentiation media (and filtering thecells to form a single cell suspension) and replating the cells on a newadherent surface in the dedifferentiation media.

In some embodiments of the invention step 1) of the method of makingDCCPCs results in differentiating the pPS cells down the chondrocytelineage pathway. Accordingly, step 1) comprises a method ofdifferentiating pPS cells into cells of the chondrocyte lineage. Cellsof the chondrocyte lineage may express one or more of the markers chosenfrom collagen II, collagen I, collagen X, aggrecan. Cells of thechondrocyte lineage may have a rounded morphology.

4. Methods of Producing Chondrocyte Lineage Cells and Chondrocytes

In some embodiments the invention provides a method for producing achondrocyte comprising 1) obtaining an DCCPC and 2) differentiating theDCCPC into a chondrocyte.

In other embodiments the invention provides a method of producing a cellexpressing one or more of the following markers: collagen II, aggrecan,and GAGs comprising 1) obtaining an DCCPC and 2) differentiating theDCCPC into a cell expressing one or more of the following markers:collagen II, aggrecan, GAGs.

In some embodiments the DCCPC may obtained by making DCCPCs according toany of the methods described infra. In other embodiments the DCCPC maybe acquired from an individual or entity that has produced it.

In some embodiments the DCCPC are differentiated into chondrocytes byculturing the cells in a chondrogenic media for a suitable length oftime. In other embodiments the DCCPC are differentiated into cellsexpressing one or more of the following markers: collagen II, aggrecan,GAGs, by culturing the cells in a chondrogenic media for a suitablelength of time.

In some embodiments of the invention the DCCPC are first grown in adedifferentiation media. To begin differentiating the cells according tostep 2) the cells are switched to a chondrogenic media. Switching themedia may include a step in which the cells are passaged, e.g. removedfrom an adherent surface with a suitable compound or combination ofcompounds such as trypsin, pelleted by centrifugation, and resuspendedin the chondrogenic media and replated on an adherent surface ormaintained in suspension as a construct of cells in the chondrogenicmedia. In other embodiments the cells are not passaged. Instead thecells remain attached to an adherent surface and the media is decanted.Optionally the cells may be washed with an appropriate buffer such asPBS and then fed with the chondrogenic media.

A chondrogenic media may comprise one or more of: linoleic acid andbovine serum albumin. The media may further comprise one or more of thefollowing: dexamethasone, insulin, transferrin, selenium, ascorbic acid,sodium pyruvate, and transforming growth factor β (TGFβ) e.g. TGFβ3. Incertain embodiments the media does not comprise an exogenously addedbone morphogenic protein. In certain embodiments the media does notcomprise a serum replacement such as knock out serum replacement.

A suitable media concentration of linoleic acid may range from about 2mg/ml to about 10 mg/ml; from about 3 mg/ml to about 7 mg/ml; from about4 mg/ml to about 6 mg/ml. In one embodiment of the invention the mediais comprised of about 5.35 mg/ml of linoleic acid. A suitable mediaconcentration of bovine serum albumin may range from about 0.5 mg/ml toabout 5 mg/ml; from about 0.8 mg/ml to about 3 mg/ml; from about 1 mg/mlto about 2 mg/ml. In one embodiment of the invention the media iscomprised of about 1.25 mg/ml of bovine serum albumin. A suitable mediaconcentration of dexamethasone may range from about 10⁻⁸M to about 10⁻⁶;from about 10⁻⁷ M to about 10⁻⁶M; from about 10⁻⁸M to about 10⁻⁷ M. Inone embodiment of the invention, the media is comprised of about 10⁻⁷ Mdexamethasone. A suitable media concentration of insulin may range fromabout 3 ng/ml to about 20 ng/ml; from about 5 ng/ml to about 15 ng/ml;from about 6 ng/ml to about 9 ng/ml. In one embodiment of the inventionthe media is comprised of about 6.25 ng/ml insulin. A suitable mediaconcentration of transferrin may range from about 3 ng/ml to about 20ng/ml; from about 5 ng/ml to about 15 ng/ml; from about 6 ng/ml to about9 ng/ml. In one embodiment of the invention the media is comprised ofabout 6.25 ng/ml transferrin. A suitable media concentration ofselenious acid may range from about 3 ng/ml to about 20 ng/ml; fromabout 5 ng/ml to about 15 ng/ml; from about 6 ng/ml to about 9 ng/ml. Inone embodiment of the invention the media is comprised of about 6.25ng/ml selenious acid. A suitable media concentration of proline mayrange from about 20 μg/ml to about to about 80 μg/ml; from about 30μg/ml to about 70 μg/ml; from about 40 μg/ml to about 60 μg/ml. In oneembodiment of the invention the is comprised of about 40 μg/ml ofproline. A suitable media concentration of ascorbic acid may range fromabout 30 μg/ml to about 90 μg/ml; from about 40 μg/ml to about 80 μg/ml;from about 45 μg/ml to about 60 μg/ml. In one embodiment of theinvention the media is comprised of about 50 μg/ml of ascorbic acid. Asuitable media concentration of TGFβ may range from about 1 ng/ml toabout 30 ng/ml; from about 5 ng/ml to about 20 ng/ml; from about 8 ng/mlto about 15 ng/ml. In one embodiment of the invention the media iscomprised of about 10 ng/ml of TGFβ.

A suitable length of time for culturing the DCCPC in a chondrogenicmedia may range from about 4-28 days; from about 5-25 days; from about7-21 days. In some embodiments the DCCPC are cultured in chondrogenicmedia for about 7 days; for about 10 days; for about 15 days; for about21 days; for about 25 days. In one embodiment the DCCPCs are cultured inchondrogenic media for 21 days.

In some embodiments of the invention the method of producingchondrocytes comprises culturing the DCCPC adherently. In someembodiments of the invention the method of producing cells expressingone or more of the following markers: collagen II, aggrecan, GAGs;comprises culturing the DCCPC adherently. In some embodiments of theinvention the DCCPC are obtained without ever forming an embryoid body.In some embodiments of the invention the cells are maintained adherentlythroughout the method (it is understood that the cells may be removedfrom the adherent surface briefly for passaging, but otherwise aremaintained adherently).

In some embodiments the method comprises a step of forming a construct.A construct may be formed by removing the cells from an adherent surfaceand pelleting the cells, e.g. by centrifugation. For example a constructof DCCPC may be formed and placed in chondrogenic media in carrying outstep 2). In other embodiments the method may not include a step offorming a construct of cells. A suitable number of cells for forming aconstruct may range from about 100,000 cells to about 600,000 cells;from about 200,000 cells to about 500,000 cells; from about 250,000cells to about 350,000 cells. In one embodiment about 250,000 cells areused to form the construct.

In some embodiments culturing DCCPC adherently may include culturingthem on a plastic tissue culture surface. In other embodiments of theinvention culturing DCCPC adherently may include culturing the DCCPC ona substrate. The substrate may comprise extra cellular matrixcomponents. In some embodiments the substrate may comprise laminin. Insome embodiments the substrate may comprise an extract from a murinesarcoma cell, e.g. Matrigel®. In some embodiments the substrate is notcollagen. In some embodiments the substrate is not gelatin. In someembodiments both step 1) and step 2) of the method are performed oncells attached to an adherent surface. In other embodiments the DCCPCare obtained from an adherent surface, but step 2) is performed whilethe cells are in suspension, e.g. in the form of a construct.

5. Methods of Administering Cell Compositions to a Subject

In certain embodiments the invention provides a method of administeringa cellular composition comprising a chondrocyte lineage cell to subjectsuch that the cellular composition engrafts in the subject withoutgenerating an immune response to the cellular composition that wouldreject the engrafted cellular composition comprising 1) obtaining acellular composition comprising chondrocyte lineage cell and 2)administering the chondrocyte lineage cell to the subject withoutadministering an immuno-modulatory compound to the subject. Suitablechondrocyte lineage cells may include for example a mature chondrocyte,an DCCPC, a cell expressing one or more of the following markers:collagen II, aggrecan, GAGs. In other embodiments immunosuppressantdrugs may, though, be co-administered subsequently, simultaneously orseparately as appropriate.

In certain embodiments the chondrocyte lineage cells are maintained as agraft without generating an immune response for about 10 days, about 30days, about 60 days, about 90 days, about 180 days, about one year. Inother embodiments the chondrocyte lineage cells are maintained as agraft without generating an immune response for more than a month, morethan 3 months, more than 6 months, more than a year.

The chondrocyte lineage cell may be administered to any site in need ofcartilage repair or restoration. As an example the cells may beadministered to an arthritic joint or a site that has suffered acuteinjury (FIG. 4). Such procedures using chondrocytes to repair apatient's knee cartilage are known in the art. For example, theCARTICEL™ autologous chondrocyte implantation (ACI) procedure involvestaking a sample of cartilage from a low weight bearing location andexpanding the explanted chondrocytes in vitro. In a later surgery aperiosteal patch is sutured to the surface of the cartilage defect andthe cultured chondrocyte preparation is injected under the patch andfilling the defect.

As described above, the chondrocyte lineage cell preparation may beadministered without an immuno-modulatory compound such as an immunosuppressant typically administered with cell grafts. Alternatively, suchcompounds may be used as appropriate. Examples of immuno-suppressantsinclude cyclosporin, FK-506 and examples of immuno-modulatory compoundsare anti-inflammatory agents such as steroidal compounds, e.g.prednisone and the like.

The administered cell composition may be allogeneic with respect to thesubject. The administered cell composition may be xenogeneic withrespect to the subject. Thus in some embodiments the cell compositionwill be a complete or partial mismatch with respect to one or morealleles of the major histocompatibility complex, such as MHC I and/orMHC II. In some embodiments the cell composition may be syngeneic withthe subject. Suitable subjects include any mammal, e.g. a mouse, a rat,a dog, a cat, a cow, a horse, a sheep, a pig, a non-human primate, ahuman.

The cell composition may be administered surgically to the site.Alternatively the cell composition may be administered to the site byinjection or through the use of arthroscopic techniques.

In certain embodiments the cell composition may range from about 1×10⁴cells to about 1×10⁸ cells. In some embodiments about 1×10⁴ cells may beadministered to the subject. In some embodiments about 1×10⁵ cells maybe administered to the subject. In some embodiments about 1×10⁶ cellsmay be administered to the subject. In other embodiments about 1×10⁷cells may be administered to the subject. In some embodiments about1×10⁸ cells may be administered to the subject.

Preferred features of the second and subsequent aspects of the inventionare as described for the first aspect mutatis mutandis

The examples that follow are illustrations not meant to limit theclaimed invention

EXAMPLES Materials and Methods

FLOW Cytometery Methods:

Cell monolayers were lifted from their substrate and disaggregated usingtrypsin (Gibco). Trypsin was removed and the cells washed in PBS andfinally resuspended in PBS with 1% FBS (Gibco). Samples were separatedequally into 5 tubes containing the following antibodies at a dilutionof 1:50.

TABLE 3 Tube Antibodies Tube 1 Negative control Tube 2 FITC Anti-humanSSEA-1 Alexa Fluor ® 647 Anti-human SSEA-3 Antibody PE Anti-humanTRA-1-81 Tube 3 Alexa Fluor ® 488 Anti-human SSEA-4 Alexa Fluor ® 647Anti-human TRA-1-60-R Tube 4 FITC Mouse IgM, λ Isotype Ctrl Antibody APCRat IgM, κ Isotype Ctrl Antibody PE Mouse IgM, κ isotype Ctrl AntibodyTube 5 Alexa Fluor ® 647 Mouse IgM, κ Isotype Ctrl Antibody AlexaFluor ® 488 Mouse IgG3 Isotype Ctrl Antibody

Cells were incubated for 30 minutes at 4° C. after which time the cellswere washed in PBS. Cells were then run on a Becton DickinsonFACSCalibur™ flow cytometer using BD FACSFIow™ sheath fluid.

Immunocytochemistry Methods:

Visualization of Tra-1-60 Expression

Cell monolayers were fixed with 4% PFA for 10 minutes and then washedwith PBS (Gibco). A blocking solution containing 10% normal goat serumand 1% BSA (Sigma) was applied for 1 hour at room temperature. Tra-1-60mouse antibody (Abcam ab16288) was applied at a dilution of 1:100 in 1%BSA and 1% normal goat serum in PBS overnight at 4° C. After removal ofthe antibody the cells were washed with PBS and incubated with antimouse secondary antibody (goat anti mouse Alexa Fluor® 488 (MolecularProbes)) at 1:200 in 0.1% BSA, 1% goat serum in PBS at room temperaturefor 1 hour. After further PBS washes with cells were stained with DAPI(Molecular Probes) at 1:1000 in PBS for 10 minutes. Cells were thenmounted in fluorescent mountant (Dako) with a coverslip and visualisedusing a Zeiss fluorescent microscope

Visualization of Nanog Expression

Cell monolayers were fixed with 4% PFA for 10 minutes and then washedwith PBS (Gibco). Cells were permeabilised with 0.4% triton X-100 for 15minutes at room temperature. A blocking solution containing 10% normalgoat serum (Sigma) was applied for 1 hour at room temperature. Nanograbbit antibody (Abcam ab21624) was applied at a dilution of 1:100 in2.5% BSA and 10% normal goat serum in PBST overnight at 4° C. Afterremoval of the antibody the cells were washed with PBS and incubatedwith anti rabbit secondary antibody (goat anti rabbit Alexa Fluor® 488(Molecular Probes)) at 1:200 in 1% BSA, 1% goat serum in PBS at roomtemperature for 1 hour. After further PBS washes with cells were stainedwith DAPI (Molecular Probes) at 1:1000 in PBS for 10 minutes. Cells werethen mounted in fluorescent mountant (Dako) with a coverslip andvisualised using a Zeiss fluorescent microscope

Visualization of Oct4 Expression

Cell monolayers were fixed with 4% PFA for 10 minutes and then washedwith PBS (Gibco). Cells were incubated with 100% ethanol for 2 minutesat room temperature. A blocking solution containing 10% normal goatserum and 1% BSA (Sigma) and 0.1% triton X-100 was applied for 1 hour atroom temperature. Oct4 mouse antibody (Santa Cruz SC-5279) was appliedat a dilution of 1:50 in 1% blocking buffer overnight at 4° C. Afterremoval of the antibody the cells were washed with PBS and incubatedwith anti mouse secondary antibody (goat anti mouse Alexa Fluor® 488(Molecular Probes)) at 1:400 in PBS at room temperature for 1 hour.After further PBS washes with cells were stained with DAPI (MolecularProbes) at 1:1000 in PBS for 10 minutes. Cells were then mounted influorescent mountant (Dako) with a coverslip and visualised using aZeiss fluorescent microscope

Visualization of Collagen Type I Expression

Cell monolayers were fixed with 4% PFA for 10 minutes and then washedwith PBS (Gibco). Dako cytomation protein block was applied for 1 hourat room temperature. Collagen type I mouse antibody (Sigma) was appliedat a dilution of 1:200 in Dako REAL antibody diluent overnight at 4° C.After removal of the antibody the cells were washed with PBS andincubated with anti mouse secondary antibody (goat anti mouse AlexaFluor® 488 (Molecular Probes)) at 1:1000 in PBS at room temperature for1 hour. After further PBS washes with cells were stained with DAPI(Molecular Probes) at 1:1000 in PBS for 10 minutes. Cells were thenmounted in fluorescent mountant (Dako) with a coverslip and visualisedusing a Zeiss fluorescent microscope

Visualization of Collagen Type II Expression

Cell monolayers were fixed with 4% PFA for 10 minutes and then washedwith PBS (Gibco). Dako cytomation protein block was applied for 1 hourat room temperature. Collagen type II mouse antibody (CII-C1 clone,University of Iowa hybridoma bank) was applied at a dilution of 1:20 inDako REAL antibody diluent overnight at 4° C. After removal of theantibody the cells were washed with PBS and incubated with anti mousesecondary antibody (goat anti mouse Alexa Fluor® 488 (Molecular Probes))at 1:1000 in PBS at room temperature for 1 hour. After further PBSwashes with cells were stained with DAPI (Molecular Probes) at 1:1000 inPBS for 10 minutes. Cells were then mounted in fluorescent mountant(Dako) with a coverslip and visualised using a Zeiss fluorescentmicroscope

Visualization of Collagen Type X Expression

Cell monolayers were fixed with 4% PFA for 10 minutes and then washedwith PBS (Gibco). Dako cytomation protein block was applied for 1 hourat room temperature. Collagen type X mouse antibody (Sigma) was appliedat a dilution of 1:20 in Dako REAL antibody diluent overnight at 4° C.After removal of the antibody the cells were washed with PBS andincubated with anti mouse secondary antibody (goat anti mouse AlexaFluor® 488 (Molecular Probes)) at 1:1000 in PBS at room temperature for1 hour. After further PBS washes with cells were stained with DAPI(Molecular Probes) at 1:1000 in PBS for 10 minutes. Cells were thenmounted in fluorescent mountant (Dako) with a coverslip and visualisedusing a Zeiss fluorescent microscope

Visualization of Cbfa1/RunX2 Expression

Cell monolayers were fixed with 4% PFA for 10 minutes and then washedwith PBST. A blocking solution containing 10% normal goat serum and 1%BSA (Sigma) and 0.1% triton X-100 was applied for 1 hour at roomtemperature. Cbfa1 rat antibody (R&D MAB2006) was applied at a dilutionof 1:200 in 1% blocking buffer overnight at 4° C. After removal of theantibody the cells were washed with PBST and incubated with anti ratsecondary antibody (goat anti rat Alexa Fluor® 488 (Molecular Probes))at 1:400 in PBS at room temperature for 1 hour. After further PBS washeswith cells were stained with DAPI (Molecular Probes) at 1:1000 in PBSfor 10 minutes. Cells were then mounted in fluorescent mountant (Dako)with a coverslip and visualised using a Zeiss fluorescent microscope

Visualization of Ki67 Expression

Cell monolayers were fixed with 4% PFA for 10 minutes and then washedwith PBS (Gibco). Cells were permeabilised with 0.25% triton X-100 for10 minutes at room temperature. A blocking solution containing 10%normal goat serum (Sigma) and 1% BSA in PBST was applied for 1 hour atroom temperature. Ki67 rabbit antibody (Abcam ab15580) was applied at adilution of 1:100 in 1% BSA in PBST overnight at 4° C. After removal ofthe antibody the cells were washed with PBS and incubated with antirabbit secondary antibody (goat anti rabbit Alexa Fluor® 488 (MolecularProbes)) at 1:200 in 1% BSA, 10% goat serum in PBS at room temperaturefor 1 hour. After further PBS washes with cells were stained with DAPI(Molecular Probes) at 1:1000 in PBS for 10 minutes. Cells were thenmounted in fluorescent mountant (Dako) with a coverslip and visualisedusing a Zeiss fluorescent microscope

Von Kossa Assay

Cell monolayers were fixed with 4% PFA for 10 minutes and then washedwith PBS (Gibco). After further washing with ddH₂O the samples wereincubated with 5% silver nitrate in ddH₂O under strong light for 1 hour.After further washes with ddH₂O the samples were incubated with 5%sodium thiosulphate for 5 minutes. After further washes in ddH₂O thesamples could be imaged on a brightfield microscope.

Visualisation of LDH Activity

Sections of tissue were made 10 μm thick on a cryostat and maintained at−20° C. until use. Polypep™ stock solution: 4% Polypep™, 0.05M gly-gly,0.017M NaOH in ddH₂O. Reaction mixture: (60 mM Lactic acid, 1.75 mg/mlNicotinamide Adenine Nucleotide, 3 mg/ml Nitroblue Tetrazolium inPolypep™ stock (all chemicals from Sigma)). Add reaction mixture to thesamples and incubate at 37° C. for 3 hours. Samples are then rinsed inddH₂O, then acetone and finally in PBS before mounting and imaging on abrightfield microscope.

Culture Media

Chondrogenic media: DMEM (Sigma D5671), 1% insulin, transferrin,selenium (6.25 ng/ml insulin, 6.25 mg transferrin, 6.25 ng/ml seleniousacid, 1.25 mg/ml bovine, serum albumin, and 5.35 mg/ml linoleic acid)(BD Biosciences 354352), 1% L-glutamine (Gibco 25036), 1% non-essentialamino acids (Gibco 11140), 1% sodium pyruvate (Sigma S8636), 350 μML-proline (Sigma P5607), 0.1 μM Dexamethasone (Calbiochem 265005), 172.7μM ascorbic acid (Sigma A8960), 10 ng/ml TGF-β3

Dedifferentiation media: DMEM (Sigma D5671), 10% FBS (Gibco 10500-064)

Osteogenic media: KnockOut DMEM (Gibco 10829), 10% FBS (Gibco10500-064), 1% L-glutamine (Gibco 25036), 1% non-essential amino acids(Gibco 11140), Beta mercaptoethanol (Gibco 31350-010), 0.1 μMDexamethasone (Calbiochem 265005), 50 μM ascorbic acid (Sigma A8960), 10mM Beta glycerophosphate (Calbiochem 35675)

Example 1 Production of Chondrocyte Progenitor Cells

The DCCPCs were produced using a protocol as follows. Initially H7 EScells were grown to 80% confluency using Matrigel® coated flasks andstandard ES culture conditions. At this point the media was replacedwith chondrogenic media. The cells remained in their original flasks andstill on Matrigel® thus reducing cost and handling. Cells were culturedin these conditions for a further 14 days with chondrogenic mediareplaced 3 times a week. On day 14 the cell layer was washed with PBSand the chondrogenic media replaced with dedifferentiation medium. Againthe cells remained in their original flasks. After a further 5 days inculture the cells showed signs of dedifferentiation and were trypsinpassaged as single cells.

Materials

The materials used were: conditioned media, bFGF (Peprotech), tissueculture flasks (Nunc), Matrigel® (BD Biosciences), DMEM (Sigma), and FBS(Gibco).

Protocol

hESCs were cultured to confluency on Matrigel® in either T25 or T75flasks under standard feeder free hESC culture conditions withconditioned media supplemented with 10 ng/ml bFGF. Then the media wasaspirated and the cells washed with PBS. Subsequently, full chondrogenicmedia was applied to the cells (DMEM (Gibco), 10⁻⁷ M Dexamethasone(Calbiochem), ITS+Premix (6.25 ng/ml insulin, 6.25 mg transferrin, 6.25ng/ml selenious acid, 1.25 mg/ml bovine serum albumin, and 5.35 mg/mllinoleic acid, BD Biosciences), 40 μg/ml L-proline (Sigma), 50 μg/mlascorbic acid (Sigma), 100 μg/ml sodium pyruvate (Sigma) and 10 ng/mlTGF-β3 (Peprotech)) Volumes used were comparable to those used inroutine culture i.e. 7 ml for T25 and 15 ml for T75.

The cells were then cultured in the chondrogenic media for 14 days withmedia changes 3 times a week. On day 14 the cells appeared condensedinto colonies surrounded by clumps of dead and floating cells. The mediawas aspirated and the cells were washed with PBS.

The media was then replaced with DMEM with 10% FBS (Gibco)(“dedifferentiation media”) and cultured for a further 5 days with mediachanges 3 times a week. During these 5 days the cells proliferated andmigrated out of the colonies such that the flask was >80% confluent byday 5. At this stage the cells are DCCPC of the invention.

On day 5 the cells were passaged with trypsin using the standard method.The vast majority of cells lifted as single cells within 5 minutes.Larger clumps of colonies were lifted from the surface by tapping theflask or pipetting the media. DMEM with FBS was added to stop thetrypsin. Cells were spun out of suspension and the trypsin supernatantremoved. Cells were passed through a 50 μm pore sized filter in order toobtain a single cell suspension in dedifferentiation media.

Cells were then plated onto plastic or Matrigel®. Alternatively cellswere made into constructs using the standard method used for the hESCs(250,000 cells in 1 ml chondrogenic media in a 15 ml tube spun at 800rpm for 5 minutes and cultured in this format for 7-21 days with mediachanges and centrifugation every 3 days. Where the construct method wasused, the cells formed a construct within 16-48 hours in chondrogenicmedia. Where the plating method was used, cells were given 24 hours indedifferentiation media to adhere to the surface before changing tochondrogenic media.

It was found that using these conditions DCCPCs plated onto plastic willmaintain cell number but will not proliferate to any degree in thededifferentiation media, i.e. they will not bulk up under theseconditions. It was also found that plastic adherent cells expressed noES or MSC markers. When Matrigel® was used as a substrate a higherplating efficiency was observed. DCCPCs on the Matrigel® surface showedevidence of proliferation when the cells were maintained inde-differentiation media. When DCCPCs were plated onto a Matrigel®substrate and maintained in EB media (Knockout™ D-MEM (Gibco), 10% FBS(Gibco), 1% L-glutamine (Gibco) 1% non-essential amino acids (Gibco) and0.1 mM Beta mercaptoethanol (Gibco)) the cells showed a high rate ofproliferation with no loss of chondrogenic potential.

Example 2 Morphology

The morphology of the cells changes dramatically throughout theprotocol. After 14 days in our chondrogenic medium the cells showclassic chondrocyte morphology with rounding up of the cell body andclustering of cells into dense colonies (FIG. 1). On day 14 the media ischanged to dedifferentiation media and at days 15 through 19 indedifferentiation media, the cells show a fibroblast like morphology andstart to repopulate the culture surface

A high proportion of the original ES cells died and formed rafts of deadcells that appeared as phase bright clumps just above the cell layer.These clumps could be washed off but not without substantial agitation.After 14 days the confluency was as low as 20%. However, the increase incolony density suggested that this decrease in confluency is notdirectly related to a decrease in cell number.

After replacement of the chondrogenic medium with de-differentiationmedia, cell morphology changed dramatically. Cells migrated out of thecolonies (as seen using time-lapse photomicrography) and took on a morefibroblast-like morphology. Cells proliferated and over the next 5 daysstarted to repopulate the flask. The rafts of dead cells also washed offduring this dedifferentiation stage such that by day 19 there were nodead cells left. At this point the cells are still in their originalflasks and thus on the original Matrigel® coating. In this state thecells proliferate. It is only after the trypsin treatment and platingonto plastic that the DCCPC stop proliferating.

The DCCPC cells had a morphology different to that of isolated primarychondrocytes. The DCCPC grew as single motile cells, and moved away fromdense cell clusters. This was very unlike the more rounded primarychondrocytes that actively form condensed 3 dimensional colonies.

Example 3 Characterisation of the DCCPCs

The de-differentiated cells were trypsin passaged and detached from theMatrigel® (and also plastic in later time points) in <5 minutes, assingle cells. At this point the suspension of cells was used either forconstruct formation or the plastic adherent cells seeded forcharacterisation.

Plastic Adherent Cells:

Plastic adherent DCCPCs maintained in de-differentiation media analysedusing FLOW cytometry showed no ES pluripotency markers or MSC markers.

In agreement with the FLOW data, immunocytochemistry using a differentTra-1-60 antibody showed no evidence of TRA-1-60 protein expression inthe DCCPCs. Tra-1-60 staining was not present on the plastic adherentDCCPCs. Undifferentiated H7 ES cells were used as a control for theantibody and showed bright membrane associated staining. After counting1053 cells no TRA-1-60 positive cells were seen in the DCCPC population.Therefore TRA-1-60 protein expression is undetectable in thispopulation.

The DCCPCs did not express the pluripotency marker Oct4 as determined byICC No evidence of Oct4 nuclear staining was seen in the DCCPCs. H7 EScells used as positive controls for the antibody show bright nuclearstaining. Cells incubated in the absence of primary antibody were usedas a negative control for the staining. Out of 1537 cells counted, noneshowed nuclear Oct4 staining.

The DCCPCs did not express the pluripotency marker Nanog as determinedby ICC. H7 ES cells used as positive controls for the antibody showbright nuclear staining. Cells incubated in the absence of primaryantibody were used as a negative control for the staining. Out of 479cells counted, none showed nuclear Nanog staining suggesting that Nanogprotein expression was undetectable in this population.

The plastic adherent DCCPCs were also analysed by ICC for collagen IIexpression, a marker of chondrogenesis, and CBFA1/RunX2 a nuclear markerfor osteogenesis and hypertrophic chondrocytes—although it is notlimited to these cell types. The majority of DCCPCs are positive forCBFA1/RunX2 (FIG. 2), unlike primary (non-hypertrophic) chondrocytesthat lack CBFA1/RunX2 protein expression. 99 out of 1482 counted cells(˜7%) showed no collagen II expression. The rest of the cells showed alow level of intracellular collagen II protein expression.

In FIG. 2, the nuclear cbfa1/RunX2 stain was found to be present in 1336out of 1575 cells. Therefore 85% of plastic adherent DCCPC show nuclearCBFA1/RunX2 protein expression.

DCCPCs in Construct Format

As mentioned previously the DCCPCs can be cultured as a construct inorder to redifferentiate the cells into chondrocytes. 250,000 cells in 1ml chondrogenic media were placed in a 15 ml tube and spun at 800 rpmfor 5 minutes and cultured in this format for 7-21 days with mediachanges and centrifugation every 3 days. Where this method was used, thecells formed a three dimensional construct within 16-48 hours inchondrogenic media. As with the plastic adherent cells, the cells withinthe construct format were also analyzed. The analyzed cells had been inconstruct format for 7, 14 and 21 days.

The growth state of the DCCPC-generated construct was investigated withantibodies raised against Ki67. Antibodies raised against this antigenwill pick up cells in all stages of the cell cycle and only those in G0will not stain. There was no evidence of nuclear Ki67 staining in any ofthe DCCPCs constructs suggesting that the cells are no longer cycling(quiescent), are not proliferative.

Pluripotency markers were investigated within the construct format byICC. No Oct4, TRA-1-60 or Nanog protein expression was found in any ofthe cell constructs be this at day 7, 14 or 21. Pelleted ES cells wereused as a positive control for these assays.

Example 4 Chondrogenic Re-Differentiation of Plastic Adherent DCCPCs

Chondrogenic Re-Differentiation:

DCCPCs were re-differentiated for a further 21 days on plastic byapplication of chondrogenic media. Collagen protein expression wasinvestigated to determine whether the cells were becoming chondrocytelike. As previously mentioned the DCCPCs show low collagen type IIprotein expression when analysed by ICC. However, when the plasticadherent DCCPCs are treated with chondrogenic media the collagen type IIprotein expression is increased. In contrast collagen type X expressionis not present throughout the DCCPC protocol and does not increase afterre-differentiation. Together this data suggests that after treatmentwith chondrogenic media the DCCPC are differentiating towards thechondrocyte lineage but are not becoming hypertrophic.

Osteopenic Re-Differentiation:

DCCPCs were analysed to see if they would differentiate into osteoblastswhen cultured in osteogenic medium. Although not osteoblast specificcollagen type I is essential for bone formation. After 21 days ofculture in osteogenic medium the cells produced extracellular collagentype I detected in ICC assays (FIG. 3). The formation of collagen type Iis known to be upregulated by primary chondrocytes that dedifferentiatewhen cultured in a monolayer format on plastic, hence the appearance ofthe protein here is not a conclusive marker of osteogenesis. Thedifferentiated DCCPCs were unable to mineralise the excreted matrix whenanalysed using a von kossa reaction. The von Kossa reaction is acceptedto be a marker of calcium ions found in mineralised tissue.Mineralisation is a requirement of bone formation and its absence hereindicates that the cells are not functional osteoblasts. As a control H1hESC directly treated with this same osteogenic medium were shown toproduce mineralised matrix. Thus this data suggests that the DCCPCsprotocol has restricted differentiation potential, indicative of adegree of lineage commitment. The protocol would seem to be limiting thedifferentiation potential of the cells to the chondrogenic lineage.

Example 5 Chondrogenic Re-Differentiation of DCCPCs in Construct Format

DCCPCs that were made into the construct format were analysed for cellviability using the lactate dehydrogenase (LDH) assay. In this assay allthe cells showed a dark stain indicative of active LDH. This datasuggests that the whole three dimensional construct is viable at days 7,14 and 21.

Primary human articular cartilage tissue is composed of a large amountof extracellular matrix such that less than 2% by volume of the maturetissue is occupied by chondrocytes (Hunziker, Quinn et al. 2002). Thecomposition of the matrix produced by the chondrocytes is important forits function, and can also be used as a marker of chondrogenicdifferentiation.

Alcian blue staining at pH 1 was used to visualise glycosamino-glycans(GAGs) other than hyaluronic acid. DCCPCs constructs showed relativelylow staining except at day 14. Safranin O staining was also used toanalyse the GAG content of the matrix produced by the DCCPC. Safranin Ostaining (which uniformly stains GAGs) was lowest in the day 7 samplesand increased slightly by day 14. There was then a very dramaticincrease at day 21. Together this data suggest that GAG content of theDCCPCs constructs increases throughout culture and that it ispredominantly an increase in hyaluronic acid and other acidic GAGs.

Collagens contribute approx 60% of the dry weight of articularcartilage. Collagen type II accounts for 90-95% of this collagencomponent. DCCPC constructs show low levels of collagen type II stainingat day 7. The level of staining increases throughout the constructthrough days 14 and 21 that correlates with the increase seen in GAGcontent of the matrix. Day 7 DCCPC constructs were also analysed forcollagens type I and type X. No collagens were observed at this earlytime point.

Constructs generated from the DCCPCs rapidly grow in size from day 14 today 21. This size increase appears to be due to matrix production ratherthan cell proliferation since the Ki67 assay shows no cells in the cellcycle. DAPI staining shows very few cells per construct volume in theday 21 section.

Example 6 Implantation of Chondrocyte Cell Constructs in an In VivoModel

Adult three months old male Sprague-dawley rats (immunocompetent wildtype; WT) (Harlan UK limited) were used in this study. All animals weremaintained under the guidelines set by the institutional Animal Care andEthical Committee at the University of Edinburgh, and the proceduresapproved under a UK Home Office license. All operations were performedunder general anaesthesia (2-3.5% isofluorane). Post-operative analgesiawas induced by buprenorphine injections (0.05% mg/Kg) twice 24 hourspost-surgery. A standard medial parapatellar approach was used to exposethe knee. A 1 mm diameter chondral defect was created in the articularcartilage in the weight-bearing area of the femoral trochlea of the ratknee. One 14 day old DCCPC construct was implanted into the defect andsealed with fibrin glue (Tisseel®, Baxter Healthcare Ltd, Newbury, UK).A layered closure was performed. No immunomodulatory drugs were used.Animals were allowed to move freely postoperative with adequateanalgesia. Samples were harvested after 3 weeks. Animals were sacrificedby inhalation of CO₂ (approved by the schedule 1 of the Animals(Scientific Procedures) Act 1986) and the dissected knees were brieflyimmersed in 5% polyvinyl alcohol (PVA; Sigma-Aldrich) and immediatelysnap-frozen in hexane (Sigma-Aldrich) maintained at −80° C.

For histological examination, frozen knees were cryosectioned (10 μm)sagittally perpendicular to the defect and mounted on histologyultra-thin tape to maintain morphology of the tissue. Cryosections werefixed in 4% (w/v) paraformaldehyde and washed in PBS. Haematoxylin &Eosin staining was used for histological analysis. For results see FIG.4.

The skilled reader will appreciate that the invention can be modified asa matter of routine optimization, without departing from the spirit ofthe invention, or the scope of the appended claims

The invention claimed is:
 1. A method for preparing chondrocyteprecursor cells, comprising the steps of: (a) providing a culture ofprimate pluripotent stem (pPS) cells in chondrogenic media, wherein thepPS cells are greater than 75% confluent; (b) differentiating the cellsof (a) by further culturing in chondrogenic media until the cells formcolonies; and (c) culturing the cells obtained in (b) inde-differentiation medium that lacks factors necessary for chondrogenicdifferentiation, until cells de-differentiate and migrate out of thecolonies, to provide a population of de-differentiated chondrocyteprecursor cells, wherein the de-differentiated chondrocyte precursorcells are characterized by the loss of transdifferentiation potential toform osteoblasts when cultured in osteogenic medium.
 2. The method ofclaim 1, wherein the population of de-differentiated chondrocyteprecursor cells obtained in (c) are characterized in that 1% or less ofthe cells express Oct4, Nanog and tra-1-60, and 85% or more of the cellsexpress CBFA
 1. 3. The method of claim 1, wherein the pPS cells arehuman embryonic stem cells.
 4. The method of claim 2, wherein the pPScells are human embryonic stem cells.
 5. The method of claim 1, whereinat least 25% of the population of de-differentiated chondrocyteprecursor cells generated in step (c) synthesize either Type II collagenor aggrecan.
 6. The method of claim 1, wherein 7% or less of thepopulation of de-differentiated chondrocyte precursor cells generated instep (c) express no collagen II.
 7. The method of claim 1, wherein 7% orless of the population of de-differentiated chondrocyte precursor cellsgenerated in step (c) express collagen X.
 8. The method of claim 1,wherein 7% or less of the population of de-differentiated chondrocyteprecursor cells generated in step (c) express CD105.
 9. The method ofclaim 1, wherein 7% or less of the population of de-differentiatedchondrocyte precursor cells generated in step (c) express Stro-1.